ADVANCES IN PROTEIN CHEMISTRY VOLUME 16
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ADVANCES IN PROTEIN CHEMISTRY EDITED BY
C. B. ANFINSEN, JR.
M. L. ANSON
Laborafory o f Cellular Physiology National Hwrf lnrtifuk Befhesda, Maryland
London, England
JOHN T. EDSALL
KENNETH BAILEY
Biological Laboratories Harvard Univeraity Cambridge, Massachusetts
University o f Cambridge Cambridge, England
VOLUME 16
1961
ACADEMIC PRESS
New york and London
COPYRIGHT @ 1961, BY ACADEMICPRESS INC. ALL RIGHTS RESERVED
NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.
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United Kingdom Edition
Published by ACADEMIC PRESS INC. (LONDON) LTD. BERKELEY SQUARE HOUSE BERKELEY SQUARE, LONDON, W.1
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PRINTED IN THE UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME 16
P. DESNUELLE, Laboratoire de Chimie, Biologique, Facultd des Sciences, Marseille, France MALCOLM DIXON,Department of Biochemistry, University of Cambridge, England PAULDOTY,Department of Chemistry, Harvard University, Cambridge, Massachusetts WILLIAM F. HARRINGTON, McCoZlum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland M. ROVERY, Laboratoire de Chimie Biologique, FacultS des Sciences, Marseille, France PETERURNES,Department of Chemistry, Harvard University, Cambridge, Massachusetts PETERH. VON HIPPEL,Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire EDWINC. WEBB,Department of Biochemistry, University of Cambridge, England B. WITKOP,Laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland JEN TsI YANG,Cardiovascular Research Institute and Department of Biochemistry, University of California Medical Center, S a n Francisco, California
V
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CONTENTS CONTRIBUTORS TO VOLUME
16... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
The Structure of Collagen and Gelatin WILLIAMF. HARRINGTON AND PETER H. VON HIPPEL
I. Introduction . . . . . . . . . . . . . . . . . . , . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . 11. Synthetic Polypeptides Related to Collagen. . . . . . . . . . . . . . . . . . . . . . . . . 111. The Composition and Amino Acid Sequence of Collagen and Gelatin.. . IV. The Structure of Collagen.. , , , , . , . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . V. The Structure of Gelatin.. . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . , . . . . , , . . . , . , . . . . . . , , . . . . . . . . . . . . . . . . . . . . . . . , . . . .
1 6 30 42
95 127
The Proteins of the Exocrine Pancreas
P. DESNUELLE AND M. ROVERY I. Introduction . . . . . . . . . . . . . . ... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biosynthesis of Pancreatic Enzymes. . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . 111. Recent Advances in the Chemical Characterization of Some Proteins of Exocrine Pancreas. . . . . . . . . . , , . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
139 141 153 191
Enzyme Fractionation by Salting-Out: A Theoretical Note MALCOLM DIXON AND EDWINC. WEBB
I. Introduction . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Theory of Salting-Out . . . . . . . . . , . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . , . . 111. Application of Theory to Protein Fractionation.. . . . . . . . . . . . . . . . . . . . . IV. Conclusions Relating to Enzyme Fractionation . . . . . . . . . . . . . . . . . . . . . . References. . . , . . . . , . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . ,
197 198 211 217 218
Nonenzymatic Methods for the Preferential and Selective Cleavage and Modification of Proteins R. WITKOP I. Introduction . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . .. . . . . . . ... .. 11. Actual and Potential Functionality of Amino Acids.. . . . . . . . . . . . . . . . . . 111. Classification of Mechanisms of Preferential and Selective Cleavages. . . IV. Principles of Selective Cleavage.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
221 224 225 237
viii
CONTENTS
V. Selected Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Induction or Prevention of Enzymatic Cleavage by Chemical Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
277 310 313 314
The Viscosity of Macromolecules in Relation to Molecular Conformation JENTSIYANG I. Introduction.. . . . . . . . . . . .
111. Newtonian Viscosities. . . .
................................
331
..................................... V. Experimental Methods. .. VI. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . .
375
References . . . . . . . . . . . . . . . .
............................
396
Optical Rotation and the Conformation of Polypeptides and Proteins PETER URNESAND PAULDOTY I. Introduction.. . . . . . . . . . . . . . . . . . . . 11. The Analysis of Rotatory Dispersio 111. The Optical Rotatory Dispersion of Synthetic Polypeptides. . . . . . . . . . . 424 ion of Proteins. . . . . . . . . . . . . . . . . . . . . . . . 481 .................................. 534 .......................................
..................................
SUBJECT INDL "X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
545
THE STRUCTURE OF COLLAGEN AND GELATIN WILLIAM F. HARRINGTON McCollum-Pratt Institute, The Johns Hopkinr University, Baltimore, Maryland
and
PETER H. VON HIPPEL Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Synthetic Polypeptides Related t o Collagen. . . . . . . . . . . . . . . . . . . A. Amino Acid Structures ...... ............. . . B. Simple Peptides of Gly roline, oxyproline.. . . . . . C. Physical Chemical Properties of Synthetic Polypeptides. . . . . . . . . 111. The Composition and Amino Acid Sequence of Collagen and Gelatin. . . . A. Amino Acid Composition.. . . . . . . . . . . . . . . . . . . . . ............... B. Amino Acid Sequence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Structure of Collagen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Structural Studies in the Solid State. . . . . . . . . . . . . . . . . . . . . . . . . . . B. Structural Studies in Solution.. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. C. The Collagen + Gelatin Transition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The Use of Proteolytic Enzymes in Structural Studies of Collagen. . , . E. The Role of Water in the Collagen Structure. ..... V. The Structure of Gelatin. ............................ A. The Molecular Properties of Gelatin a t Room Temperature and Above. B. The Molecular Properties of Gelatin a t Low T References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............
1 6
7 ‘3
11 30 30 35 42 42 64
75 83
96
127
I. INTRODUCTION The properties of collagen are of interest from many points of view. Since it constitutes the major protein component of skin, bone, tendon, and all the other forms of connective tissue, an understanding of collagen seems to the clinician to be a necessary (though not sufficient) prerequisitc to a rational attack on the many and diverse connective tissue disorders currently lumped together as “collagen diseases.” The mechanisms of the biosynthesis and incorporation into collagen of the unusual amino acids hydroxylysine and hydroxyproline offer a continuing challenge to the “pathway” biochemist. The leather chemist strives for a bettcr understanding of the interaction of collagen with tanning agents, for such undcrstanding lies directly on the road to improved leather products. Thc
2
HAREINGTON AND VON HIPPEL
comparative biologist is intrigued by the direct correlation between the temperature a t which the collagen of a given organism undergoes thermal “denaturation” and the temperature of the normal habitat of that organism. And finally, collagen interests the physical biochemist, not only because of its intrinsic importance, but also because the properties of collagen on the molecular level truly belong in a class set apart from those of all other protein molecules. Clearly, it is impossible to consider, even briefly, all these facets of the collagen problem. So while recognizing both the importance and the intrinsic interest of other areas, limits of space, patience, and competence all have served to confine this review primarily to the last of the points of view mentioned above. And even this area-the molecular properties of collagen-is so large and developing so rapidly that no pretence to completeness of coverage can be made. Thus it will be apparent that this review represents an attempt to summarize the present position as seen by the authors, for although we have tried to take a balanced view throughout, some aspects of certain of the problems discussed are controversial and might have been handled quite differently by others in terms of emphasis, coverage, and perhaps interpretation. However, despite these difficulties, we hope that we have succeeded in assembling a reasonably complete and coherent account of the current situation. Since “The Structure of Collagen Fibrils” was last reviewed in these pages by Bear (1952), tremendous progress has been made in the field of protein chemistry and structure analysis. And this progress has naturally been accompanied by comparable progress in our understanding of the collagen molecule. The magnitude of this development in the field of collagen might best be indicated by summarizing some of the major drvelopments of the decade which has passed since Rear wrote his review in the spring of 1951. 1. It had been clear for many years prior to 1951 that collagen fibers from all organisms have in common a distinctive wide-angle X-ray diffraction pattern, and thus presumably also a unique polypeptide chain configuration. However the nature of this configuration was not known. Since that time advances in our understanding of the stereochemistry of polypeptide chains and the theory of fiber X-ray diffraction first made possible, and then led to, a generally accepted structural interpretation of the wideangle X-ray pattern of collagen. 2. The unusual amino acid composition of collagen had also been recognized for some time. One-third of the residues of all collagens seemed to be glycine, while about one-fourth were proline and hydroxyproline. However, the stereochemical consequences of the presence of these residues has only become clear as a result of detailed studies of synthetic homo- and
STRUCTURE OF COLLAGEN AND GELATIN
3
copolymers of glycine and proline. Consideration of such synthetic polypeptides as simplified models of certain features of collagen and gelatin has been extremely helpful in recent years, and constitutes the rationale for the inclusion of a section dealing specifically with these synthetic polypeptides in this review. 3. Physicochemical studies of soluble collagen, which had just been initiated in 1951, have been pursued vigorously during the last 10 years and have added immeasurably to our understanding of the collagen system. Such studies, in turn, have made it possible to interpret the long-range periodicities which had previously been observed in collagen fibers using electron microscopy and small-angle X-ray diffraction, and have led to a partial understanding of the molecular basis of the patterns observed in the various “reconstituted” collagens. 4. The collagen gelatin transformation in solution has been recognized as a reversible first-order phase transition, subject to the same physical laws which govern the crystalline S amorphous phase transitions observed in systems of linear polymers. The direct relationskip between the transition in solution and the well-known thermal shrinkage phenomenon exhibited by collagen fibers has also been established. 5 . It has become clear that gelatin, long considered to be a degradation product of uncertain composition and properties, can be prepared from collagen under conditions which result in the production of perfectly reproducible protein systems. Thus gelatin itself, and the mechanism of the process by which it undergoes spontaneous reversion to a collagen-like structure on cooling, have been the object of considerable study over the last few years. Specifically, it is the recent developments in these areas which we propose to discuss in this review, For detailed considerations of earlier work and for coverage of certain peripheral topics the reader is referred to the reviews by Bear (1952) and Kendrew (1954), to the informative monographs by K. H. Gustavson entitled “The Chemistry and Reactivity of Collagen,” and “The Chemistry of Tanning Processes” (Academic Press, 1956);and to numerous recent symposia dealing with various aspects of the collagen problem, including: “Nature and Structure of Collagen” (J. T. Randall, ed., Academic Press, 1953a); “Fibrous Proteins and Their Biological Significance” (SOC.Ezptl. Bid. 9, 127 (1955) ; “Connective Tissue” (R. E. Tunbridge, ed., Blackwell, 1957); “Recent Advances in Gelatin and Glue Research” (G. Stainsby, ed., Pergamon Press, 1958); “Calcification in Biological Systems” (R. F. Sognnaes, ed., Am. Assoc. Advancement Sci., 1960); and “Central Leather Research Institute Symposium on Collagen” (N. Ramanathan, ed., Interscience Publishers, 1961). Before plunging into the detailed discussion, it is important to consider
4
HARRINGTON AND VON HIPPEL
the problem of the identification of collagen and its distribution through the “animal kingdom,” and simultaneously to establish an operational definition of this class of proteins. These problems have been treated at great 1engt)h by Bear (1952), and since recent work has only served to subshntiate most of his conclusions we will confine ourselves to a brief summary of current views. Collagen fibers may often be recognized histologically on the basis of one or more of the following bulk characteristics: they tend to swell markedly when immersed in acid, alkali, or concentrated solutions of certain neutral salts and nonelectrolytes; they are generally relatively inelastic,; they are more resistant than most protein fibers to degradation by proteolytic enzymes, but (in contrast to all other proteins studied) are readily attacked by the enzyme collagenase; they undergo thermal shrinkage to a fraction of their original length a t a temperature which is characteristic of the collagen from a given animal (but varies from one species to another) ; and they are converted in large part to soluble gelatin by prolonged treatment at temperatures above the thermal shrinkage level. I n practice, however, these criteria are not always easy to apply, nor are they infallible. Thus in recent years the presence of the characteristic collagen wide-angle X-ray diffraction pattern has come to be accepted a s the fundamental defining criterion for collagen. This pattern, (which will be discussed and interpreted in detail in Section IV) is easily recognized by the strong 2.86 A meridional spacing and by the -11 A hydration-sensitive reflection on the equator (see Fig. 8). It has served to demonstrate the presence of collagen in the tissues of almost all multicellular animals which have been investigated, ranging from the primitive porifera and coelenterates, through the annelids and echinoderms, and u p to the vertebrates (see Marks et al., 1949; Bear, 1952; Gross et al., 1956). The following physical and chemical features also seem to be generally characteristic of collagen (see Gross et al., 1958; Watson and Silvester, 1059; Piez and Gross, 1959; Piez and Likins, 1960): (1) a content of glycyl residues close to one-third of the total number present; (2) a high content of pyrrolidine-ring-containing residues (relative to the composition of most other proteins) ; (3) substantial numbers of hydroxyprolyl residues; (4) few or no cystyl, methionyl, valyl, phenylalanyl, tyrosyl, or histidyl residues, and thus minimal absorption a t 280 mp; (5) hydroxylysyl residues; (6) a n extensive meridional small-angle X-ray diffraction pattern; (7) fibrils exhibiting periodically-banded (-640 A) structure in the electron microscope; and (8) an infrared absorption band a t 3330 cm-’. In the following sections these criteria will be considered in detail, mostly in terms of certain vertebrate collagens which have been extensively studied. However, the available evidence suggests that in main outline the
STRUCTURE OF COLLAGEN AND GELATIN
w
a
results obtained with these collagens apply equally well to most vertebrate (and perhaps invertebrate) collagens. Very recently careful studies of invertebrate collagens have been launched by several groups, and it has become apparent that some of these collagens differ substantially from vertebrate collagen in certain respects. For example, the -640 A macroperiod seen via electron microscopy or small-angle X-ray diffraction in vertebrate fibers has not been detected in certain of the invertebrate materials examined. Also, while in vertebrate collagens the ratio of proline to hydroxyproline is generally in the neighborhood of unity, in the invertebrates this ratio varies widely. Thus gelatin from earthworm (Lumbricus) cuticle contains 13 prolyl and 165 hydroxyprolyl residues per 1000, while gelatin from Ascuris cuticle contains 280 prolyl and only 24 hydroxyprolyl residues per 1000 (Watson and Silvester, 1959). It is expected that careful physicochemical studies of such invertebrate collagens will help to illuminate further the role of these residues in the collagen structure. Finally, it must be mentioned that there exist certain classes of protciiis which yield the collagen wide-angle X-ray diffraction pattern and thus meet the minimum requirements for membership in the collagen group, but which differ significantly from the better known collagens in other ways. Despite previous controversy, all the following now seem securely established as collagens. (a) Reticulin: This substance is found closely associated with collagen in the connective tissue. It is identified histologically through the presence of branching networks (see Bear, 1952; Kramer and Little, 1953; Kendrew, 1954; Robb-Smith, 1957, 1958). (b) Ichthyocol and elastoidin: These terms simply refer to collagens prepared, respectively, from the swim bladders and skins of fish, and from the fins of the shark. Certain of the former, particularly the collagen derived from the swim bladder of the carp, have been extensively studied and are discussed in detail in subsequent sections. Elastoidin has been examined much less extensively (see particularly Bear, 1952; Gross and Dumsha, 1958). (c) Vitrosin: A fibrous protein obtained from the vitreous humor of the eye. It exhibits the collagen wide-angle X-ray pattern, -640 A periodicity and a typical collagen amino acid composition (see Gross et al., 1955b). (d) Spongin, gorgonin, and cornein : Fibrous proteins derived from sponges, corals, and coelenterates (see especially Marks et al., 1949; Bear, 1952; Gross et al., 1956; Pie2 and Gross, 1959). (e) The “secreted” collagens: These may be differentiated from other collagens in being secreted by epithelial cells instead of being mesodermal or mesogleal in origin. The secreted collagens show the typical wide-angle X-ray pattern, but apparently no macro-period. Examples include : earth-
6
HARRINGTON AND VON HIPPEL
worm and Ascaris cuticle, the bivalve byssus threads, and the “ejected filaments” of the sea cucumber (see Bear, 1952; Kendrew, 1954; Watson and Silvester, 1959). Despite earlier claims to the contrary, it now seems relatively well established that elastin, the third major histological component of vertebrate connective tissue (with collagen and reticulin) is not a member of the collagen class of proteins (Bear, 1952; Kendrew, 1954).
11. SYNTHETIC POLYPEPTIDES RELATED TO COLLACSN I n this section we propose to consider the effects of proline, hydroxyproline, and glycine residues on the molecular architecture of polypeptide chains. The physical chemistry of synthetic polypeptides containing these residues will be examined in detail because of the remarkable similarities which exist between the helical structures generated from polymers of these amino acids and the three-dimensional pattern of the polypeptide chains of the collagen molecule. Moreover, the mechanism of the reversion of the component chains of collagen to form the parent collagen molecule a t low temperatures in vitro (and possibly in the connective tissue in vivo) is now thought to be intimately related to the configurational changes exhibited by these model substances in solution. Pauling (1940), in considering the general problem of protein configuration, first pointed out that the insertion of a proline residue into a polypeptide chain has marked effects on the chain direction. Indeed, he demonstrated that proline residues, by virtue of their unusual stereochemistry , are ideally suited to serve as hinge points for chain folding in globular proteins. More recently, it has also become apparent that the geometry of the pyrrolidine ring prevents L-proline from being accommodated in a lefthanded a-helix. Furthermore, this residue can only fit into an undistorted right-handed a-helix when it occurs as one of the first three residues a t the N-terminal end of the chain. On the other hand, Lindley (1955) has showii that a change in helical sense (from a left-handed to a right-handed ahelix) can occur without distortion a t a proline residue. I n all cases the direction of an a-helix is altered at a proline residue and may be completely inverted if the atomic grouping about the imide linkage is disposed in the cis-configuration (Edsall, 1954). In addition to the stereochemical features imposed on a polypeptide chain as a consequence of the insertion of a proline residue, the imino nitrogen atom of the pyrrolidine ring is devoid of a proton and is consequently unable to participate in the formation of inter- or intrachain hydrogen bonds. This means that the regularity of any systematic, hydrogen-bonded peptide structure will be interrupted in proline-containing regions. The effect of such internal breaks on the stability of the a-helix has been considered by Schellman (1955) who demonstrated that usu-
STRUCTURE OF COLLAGEN AND GELATIN
7
ally a t least three hydrogen bonds would be ruptured at such a discontinuity, although little configurational entropy should be acquired by the chain since peptide units on either side of the break are maintained by hydrogen bonds. On the other hand it would be expected that insertion of an appreciable number of randomly distributed proline residues would lead to destabilization of the helix. Some evidence which supports this view comes from the work of Szent-Gyorgyi and Cohen (1957) who compared the a-helical content of a number of the keratin-myosin-epidermin-fibrinogen (KMEI') rlass proteins, as deduced from optical rotatory dispersion, with their proline content. They found that proteins with less than 30 proline residues per 1000 were in general highly folded, and that as the proline content increased above this level a polypeptide chain without benefit of stabilizing cross-bridges seemed to approach a random-coil-type configuration. At very high concentrations of proline a completely new type of configurational pattern seemed to emerge. This configuration, termed poly-L-proline 11, will be the subject of detailed consideration below. Before embarking upon the discussion of the relatively complex architecture of the synthetic polymers and copolymers related to collagen and gelatin, it is important to examine the structure of glycine, proline, hydroxyproline, and simple peptides composed of these residues. As indicated in the introduction, the stereochemistry of these residues is crucial to all current theories of collagen structure. Their importance may be judged, without reference to a particular structure, when it is recognized that, together, they comprise over 50 70of the amino acid content of most collagens.
A , A mino Acid Structures 1. Glycine
The crystal structure of glyoine has been redetermined recently by Marsh (1 9.58) from a complete three-dimensional Fourier analysis involving 1867 reflections. In general, the bond lengths and bond angles deduced were essentially identical to those reported earlier by Albrecht and Corey (1939) except that the C-N bond distance is 1.474 A instead of 1.39 A. The former distance is in good agreement with that normally observed in other amino acids. Carbon atoms 1 and 2 and oxygen atoms 1 and 2 (C, , CZ, 0, , and 0 2 ) * are nearly coplanar (see Fig. 1) whereas the nitrogen atom is 0.44 A out of plane. 2. L-Praline
A complete X-ray diffraction study of this imino acid has been prevented as a result of its strongly hygroscopic character. However, Mathieson and
* The subscript numbers with symbols here indicate position of atom in the molecule.
8
HARRINGTON AND VON HIPPEL
Welsh (1952) have deduced structure parameters from a careful investigation of copper-DL-proline dihydrate. In Fig. 1, atoms CI and N, CZ, CS, and Cg of the pyrrolidine ring are coplanar. C4 lies at a distance of about 0.60 A out of plane while the carboxyl group attached to atom Cz is disposed on the opposite side of the plane with respect to C4 . Bond angles N-C6-C4
-
L Pro1i ne
L - Hydroxyproline FIG.1. Interatomic bond lengths and bond angles for glycine (Marsh, 1958) L-proline (Mathieson and Welsh, 1952), and L-hydroxyproline (Donahue and Trueblood, 1952).
and C2-C3-Ca have the unusually low values of 96" and 97", respectively. These values may be in error since in L-hydroxyproline (Donahue and Trueblood, 1952), in L-lcucyl-L-prolylglycine (Leung and Marsh, 1957), and in poly-L-proline I1 (Cowan and McGavin, 1955; Sasisekharan, 1959a) the corresponding angles vary between 103" and 108". 3. L-Hydroxyproline The crystal structure of L-hydroxyproline was first reported by Zussman (1951) and a complete three-dimensional Fourier analysis published a year
STRUCTURE O F COLLAGEN AND GELATIN
9
later by Donahue and Trueblood (1952). As in copper-DL-prolinedihydrate, the five-membered ring is appreciably puckered. C 4 ,the carbon atom bearing the hydroxyl group, is about 0.40 A from a plane defined b y the other four atoms of the ring and lies on the opposite side of this plane from the and carboxyl group. The angle between the two planes C6-N-Cz-C3 ca-C4-c6 is 17". Neuberger's (1948) prediction that the carboxyl and hydroxyl groups should be in the trans-configuration relative to the ring has been completely confirmed by the structure of Donahue and Trueblood.
B. Simple Peptides of Glycine, Proline, and Hydroxyproline 1. Glycylglycine
Glycylglycine can exist in three different crystal forms, a,0, and y, which were originally observed by Bernal (1931) growing side-by-side in the same mother liquor. The dimensions of this simplest of all linear peptides were first determined by Hughes and Moore (1949) from a two-dimensional Patterson projection analysis of the @-form. I n this structure the heavy atoms are all coplanar except for the terminal nitrogen which is 0.64 A out of the molecular plane. More recently Hughes et al. (1954) have carried out a three-dimensional analysis of a-glycylglycine involving more than 2000 X-ray reflections. Again the whole molecule is planar except for the terminal nitrogen atom which lies 0.73 A below the plane. I n this structure the peptide bond length is 1.32 A, which is closer to the value usually observed for this bond (Corey and Pauling, 1953) than is the 1.29 A distance reported for @-glycylglycine.Both forms of the structure are shown in Fig. 2. 2. L-Leucyl-L-prolylglycine A study of the stereochemistry of this peptide is of great value in gaining an understanding of the configurational effect of a proline residue on the relative arrangement of neighboring amino acid residues. The crystal structure was determined by Leung and Marsh (1957) from a thorough three-dimensional diffraction analysis utilizing about 1700 reflections. Atoms C4, C7, C, , Cs, Nz , and 0 4 of the leucyl-prolyl peptide group are coplanar within 0.02 A, with bond lengths and angles close to those reported for other peptides (Corey and Pauling, 1953) except for the bond angle N2-Cg-4~ , which is about 5" larger than expected (see Fig. 3). Dimensions of the pyrrolidine ring are closely similar to those observed for L-hydroxy-proline (Donahue and Trueblood, 1952) with atoms C 4 ,Cg , C7 , and N Pcoplanar, and Caout of plane by 0.37 A. The dihedral angle between the plane of the leucyl-prolyl amide grouping and that of the proline ring is approximately 7". Several noteworthy features emerge from an examination of the structure
10
HARRINGTON AND VON HIPPEL
which are of special interest to the present review. Most prominent among these is the finding that the
//
0
C-N Bond ( i )
bond length is close to 1.34 A. This value is normal for a peptide bond, demonstrating that the imide linkage between the carbonyl group (C,) and the imino nitrogen (Nz) of the pyrrolidine ring possesses virtually the same degree of double bond character as do ot,her peptide linkages. Moreover thc
a - Glycylglycine
,9-Glycylglycine FIG.2. Interatomic bond lengths and bond angles for wglyclglycine (Hughee and Moore, 1949) and 8-glycylglycine (Hughes, et al., 1951).
angle CK--N~-C, of 113" is close to the value accepted by Corey and Pauling (1953) for the C,-NH angle in a peptide group. Another prominent feature is the disposition of the major groups. It appears that the leucylprolyl-glycine residues are maximally extended in the crystal structure, with the nine main-chain atoms of the proline and glycine residues (Ci-C*, N1, and 01-03) essentially coplanar. Each C,-N bond is virtually cis to the C--0 bond of the same residue. At the position of the proline residue the chain undergoes a twist of about 120"from the fully extended configuration, a twist required by the geometry of the pyrrolidine ring. 3. Tosyl-L-pro1 yl-L-h ydroxyproline Monohydrate
Only a partial three-dimensional analysis of this peptide has been reported (Beecham et al., 1958) although the known occurrence of proline-
STItUCTURE OF COLLAGEN AND GELATIN
11
hydroxyproliiie sequence in the chains of collagen (see Section 111)endow it with special significance. The molecule appears to be composed of four approximately planar groups: p-methyl-thiophenyl, prolyl, peptide pyrrolidine, and carbonyl. These are disposed at right angles to each other. Evidence of the flexibility of the pyrrolidine ring come's from the position of the carbon atom opposite the C,-N bond of the proline residue. In this structure, unlike that of proline (Mathieson and Welsh, 1952) and hy-
+
1.51
12
L-
ti I
Leucyl - L-Prolylglycine
FIG.3. Interatomic bond lengths and bond angles for ~-leueyl-~-prolylglyci~~e (Leung and Marsh, 1957).
droxyproline (Donahue and Trueblood, 1952) this carbon atom appears to be on the same side of the ring plane as the peptide C=O. Another example of the apparent flexibility of the pyrrolidine ring will be given in the discussion of the poly-L-proline I1 structure.
C . Physical Chemical Properties of Synthetic Polypeptides 1. Polyglycine II
a. Studies on Polyglycine II in the Solid State. In 1934, Meyer and Go reported an X-ray diffraction study of various polyglycine preparations, in
12
HARRINGTON AND VON HIPPEL
which it was shown that this substance can exist in two distinct and reproducible isomeric forms. When polyglycine is cast into films from dichloroacetic or trifluoroacetic acid, X-ray diffraction patterns exhibit a strong 4.4 A spot and a somewhat more intense 3.45 A reflection (form I). However, if the polymer is precipitated from concentrated aqueous lithium bromide or calcium chloride solutions, X-ray diffraction powder diagrams show a strong 4.15 A reflection (form 11).
0
5A
FIG.4a. Structureof polyglycine I1 (from Crick andRich, 1955). A projection down the screw axis, showing seven chains. Hydrogen bonds, shown as dashed lines, run in a number of directions linking neighboring chains.
X-ray evidence suggests that polyglycine I exists in the solid state as an extended @-structure (Astbury, et al., 1948; Astbury, 1949; Corey and Pauling, 1953) the two reflections of 3.45 A and 4.4 A corresponding, respectively, to the side chain and backbone spacing generally observed for this structural pattern (Bamford et aE., 1956). Additional supporting evidence comes from infrared absorption and dichroism studies of a partially oriented specimen of polyglycine I (Elliott and Malcolm, 1956) as well as a strong 1.16 A X-ray meridional reflection (Bamford et al., 1955) which is also found in @-poly-L-alanine. Assignment of the polyglycine I1 structure proved to be more dificult. Neither the very inteuse 4.15 A rcflectioii nor the infrared absorption bands
STRUCTURE OF COLLAGEN AND GELATIN
13
observed at 1648 cm-' (C = 0 stretch) and 1558 cm-I appeared to fit any known systematic configuration such as the a-helix or the 0-structures. Bamford et al. (1956) were thus led to postulate the existence of a new but unknown folded structure for this form. Readers of Nature had little time
FIG.4b. Structure of polyglycine I1 (from Crick and Rich, 1955). A projection of the structure with the screw axis vertical. The chain on the right is nearer the reader than that on the left. The planar peptide groups are edge-on a t the bottom of the figure with hydrogen bonds from these groups virtually perpendicular to the plane of the paper.
to ponder over this anomaly before a solution was offered by Crick and Rich (1955). In the polyglycine I1 structure proposed by Crick and Rich, all of the polyglycine chains are parallel and are packed in an hexagonal array. Each chain has a threefold screw axis and is hydrogen bonded t o each of its six neighbors as shown in Fig. 4a, which is a projection down the screw axis. The hydrogen bonds are linear with an 0 * N distance of 2.76 A, which is within the range of values found for simple compounds. Figure 4b presents a view normal to the screw axis where it will be seen
-
14
IlARRINGTON AND VON HIPPEL
that the planar peptide group is inclined at about 35", with its plane perpendicular to that of the paper. Movement along any chain to the peptide group of the next residue involves a rotation of 120" about, the fiber axis and a displacement in the fiber direction of 3.1 A. There are thus three residues per complete turn of the helix with a repeat distance of 9.3 A. Left-handed or right-handed helices are equally probable, since the residues of polyglycine are devoid of an asymmetric carbon atom. Moreover, for a given set of left-handed or right-handed polyglycine screws in the form I1 configuration, it appears possible to remove a chain from the structure and to replace it running in the opposite direction (Crick and Rich, 1955). Coordinates for the polyglycine I1 structure are given in Table I. TABLEI Atomic Coordinates for the Polyglycine IIa and Poly-L-proline I10 Helices (A)
Atom
Or-C C' 0 N Or41
(A)
Glycine
Proline
0 1.16 1.32 1.93 3.1
0.00 1.16 1.33 1.94 3.12 3.44 2.78 1.70
B-C1 r-C1 S-C1
~~
4)O
Glycine Proline 1.27 0.28 1.17 1.01 1.27
1.25 0.27 1.18 1.01 1.25 2.G5 3.19 2.47 ~~
-
Glycine
Proline
0 -10.5 -111.5 75 120
0.00 20"30' 112"30' -75'30' - 120"00' - 105"30' -81"OO' -64"30' ~
Crick and Rich (1955). Coordinates are for right-handed polyglycine I1 helix. b Sasisekharan (1959a).
4
b. Studies on Polyglycine II in Solution. I n solution, polyglycine exhibits rather interesting form I $ form I1 transitions, which were first observed and reported by Meggy and Sikorski (1956). Whcii polyglycirie I is dissolved in saturated aqueous calcium chloride and precipitated by dilution with water at 20"C, form I1 of the polymer results. This material appears as thin hexagonal plates in the electron microscope. If the precipitation is carried out a t 100°C, only form I is obtained and electron micrographs of this material reveal flat parallelograms. At temperatures between 60" and 100°C precipitation gives a product with an X-ray diffraction pattern intermediate between that of form I and form 11, while below 60°C only form I1 is obtained. 2. Poly-L-proline The successful synthesis of poly-L-proline by Rerger d al. (1954a) has provided one of the most, interesting and important model compounds which
STRUCTURE OF COLLAGEN AND GELATIN
15
we have for an understanding of the chemistry of collagen and gelatin. Earlier attempts to synthesize this substance were unsuccessful (Astbury et al., 1948) possibly as a result of the difficulties involved in the preparation of a highly purified N-carboxyproline anhydride, which is crucial to the synthesis. The early preparations of poly-L-proline (polymerized in bulk or in dioxane as solvent) were of relatively low molecular weight with an average degree of polymerization (DP) of 35 to 133 as determined by end-group titration (Sela and Berger, 1955). These were quite water-soluble, in contrast to the insolubility in water of poly-a-amino acids such as polyglycine, poly-L-alanine, poly-L-leucine, and poly-L-phenylalanine. With respect to its solubility properties, low molecular weight poly-L-proline resembles polysarcosine, in which the amide linkages are also devoid of a hydrogen atom (Wessely and Sigmund, 1926; Waley and Watson, 1949; Hanby et al., 1950). More recently Blout and Fasman (1958) have succeeded in obtaining very high molecular weight polyproline samples (DP = 567 to 928) via polymerization in acetonitrile. These polymers proved to be insoluble in water, Soon after the synthesis of poly-L-proline, Kurtz et al. (1958a) observed striking changes in the optical rotatory properties when aqueous or organic acid solutions of the polymers were allowed to stand at room temperature. Precipitation of the polymer from thc reaction medium with ether, followed by dissolution in water or acetic acid, resulted in a dextrorotatory solution with [a]z5= $40". On standing, the rotation slowly changed t o a highly levorotatory form with [a]E5= -420" (this value could be increased to [a]E5= -5540" by heating). The rate of mutarotation is strongly dependent on the solvent. In formic acid, mutarotation of form I ([a]E5= +40°) -+ form I1 ([a]E5= -540") is complete in less than 1 hr at 25"C, whereas in acetic acid or water a t this temperature rotatory changes are observed over a period of several days. Poly-L-proline I1 in the solid state was obtained by dissolution of form I in hot glacial acetic acid or inm-cresol a t 100" C, followed by ether precipitation after cooling. This material is readily soluble in water, acetic, or formic acids, giving a specific rotation, [a]E5= -540°, which is invariant with time. The high molecular weight samples of poly-L-proline synthesized by Blout and Fasman (1958) are also quite water-soluble after transformation to form 11. a. Studies on Poly-L-proline in the Solid State. (i) X-ray difraction analysis of poly-L-proline 11. In 1955, a structure for poly-L-proline I1 in the solid state was proposed by Cowan and McGavin, on the basis of X-ray diffraction patterns obtained from powders and oriented films. An intriguing feature of the proposed structure is the essential identity of the backbone
16
HARRINGTON AND VON HIPPEL
configuration with that of polyglycine 11. Thus it seems likely that there exist a t least three distinct classes of polypeptide chain configurations: the a-helix (Pauling and Corey, 1951a), the @-structures (Pauling and Corey, 1951b), and the recently “discovered” poly-L-proline I1 or polyglycine II-type helix. In the poly-L-proline I1 structure of Cowan and McGavin, the peptide grouping is planar and disposed in the trans-configuration. Movement along the chain from one residue to the next involves a rotation of - 120” about the fiber axis and a translation along the axis of 3.12 A. The axial repeat is therefore 9.36 A (see Fig. 5). Unlike the polyglycine I1 helix, the poly-Lproline I1 helix must be left-handed, since the screw sense is uniquely determined by the absolute configuration of groups around the a-carbon atom (Bijvoet et al., 1951). A right-handed helix of the required dimensions cannot be constructed with L-amino acids. This property is fundamental to the behavior and chemistry of the chains of collagen as will be evident in subsequent discussion. Sasisekharan (1959a) has recently published a more detailed X-ray diffraction study of powders and films of poly-L-proline 11, including an analysis of optical diffraction data. This elegant study confirms in all essential details the structure proposed by Cowan and McGavin. The bond lengths and bond angles of the pyrrolidine ring appear to be in good agreement with those deduced for L-hydroxyproline by Donahue and Trueblood (1952) and for L-leucyl-L-prolylglycine by Leung and Marsh (1957) with one interesting exception : in the poly-L-proline I1 structure all of the heavy atoms of the proline ring are virtually coplanar. Otherwise, too much strain is introduced in packing the chains into the required unit cell dimensions. The pyrrolidine rings of copper-DL-proline dihydrate, L-hydroxyproline, and L-leucyl-L-prolylglycine are all appreciably puckered, with atom Cd lying 0.40-0.60 A out of the molecular plane. It will be recalled that some flexibility in the proline ring has also been suggested for the proposed tosyl-L-prolyl-L-hydroxyprolinemonohydrate structure (Beecham et al., 1958). (ii) X-ray diffraction analysis of poly-L-proline I . The spatial geometry of poly-L-proline I in the solid state has not been rigorously established up to the present time, since no one has succeeded in obtaining oriented films or fibers of this substance. Cowan and Burge (1958) have reported preliminary X-ray and electron diffraction analyses of powder patterns which are compatible with a right-handed helix consisting of cis-prolyl residues with three residues per turn and a pitch of 6.30 A, Rich and Crick (1959) have also attempted X-ray diffraction analyses of form I powders and have suggested a similar right-handed helix of residues disposed in the cisconfiguration, with, however, three and one-eighth residues per turn and an axial repeat of 5.85 .A.
STRUCTURE O F COLLAGEN AND GELATIN
17
(iii) Infrared spectra of poly-L-proline. Infrared spectra of poly-L-proline I and poly-L-proline I1 have been reported by Berger et al. (1954b) and Blout and Fasman (1958). Both polymers show several common ab-
FIG.5. Structure of poly-L-proline. The figure on the right is a wire model of a left-handed helix having all peptide bonds in the trans-configuration. This is the structure proposed by Cowan and McGavin (1955) for poly-L-proline I1 in the solid state. Neighboring ring planes are essentially perpendicular t o each other. The figure on the left is a right-handed helix with all peptide bonds in the cis-configuration (see text).
sorption bands, notably at 2950 and 2860 cm-' (C-H stretching modes), 1650 cm-' (C = 0 stretch), and 1485 cm-' (possibly C-H vibrations of the pyrrolidine ring). At frequencies lower than 1400 cn-', the absorption spectra differ markedly. The spectrum of form I exhibits strong bands a t
18
RAILRINGTON A N D VON HIPPEL
980 and 1355 cm-’, whcreas thcse arc abscrit in form TI. One rather striking feature is a w r y strong band at 3480 cm-I, which ocrurs in the spcctra of hoth polymers (Rergcr et al., 1954a; Rlout and Ihsman, 1958). On thorough drying of thr polymcr this hand disappcars and Rlout arid Icasman conc2ludt.d that it is duc to very strongly adsorhrd watcr. It is of intmcst in this c.oiincc*tioiito tiotc that Rradhury ct al. (ln.58) havr obscrved a similar h i d in cwllagcri at 3450 cm-l, arid have demonstrated that it arises cxclusively from adsortwd water by following rhsngcs in infrared absorption as a func~tionof humidity. Polarized infrared spcctra of *form I arid form 11 reveal pcrperidicular dichroism of thc (”=() stretcahing frcqucnry a t 1650 (m-’, suggesting that thc varlioiiyl groups extcnd away from thc main chain axis (see Fig. 5). From the dirhroic ratios of -1.7 (form 11) and ~ 1 . (form 4 I) it may tie inferrrd that thc C=O groups of form I1 arc disposed more normal to the t)ac*kbonerhain than arc thosc of form I. .4 similar dichroism of the C=O band has l m i i rcportcd for oricntcd c~ollagenpreparations (Badger and l’ullin, 1954; Sutherlaiid et al., 19.54) and oricmted c.old-rvaporated gclatin filnis (Xmbroso and IClliott, 1‘351a). 0. Studics on I ’oly-i,-prolirLe in Solution. (i) The mutarotation of poly-Lprolinp. Thc mcchariisni of the mutarotation of poly-L-prolinc in solution has engagcd the attcntion of a numbrr of lahoratorics. 111thcir carly work Kurtz ct al. (19.56) suggcsted that thc change in optiral rotation whirh occurs during thc transition from form I form I1 might rcsult from a srries of cis -+ trans-trnrisformatioiis of thc prptide bond groupings in e w h molcculf.. In latcr studics, Stcinherg (1958) and Stcinherg et al. (1958) discovrrcd that poly-L-proline I1 cwuld he rcconverted to poly-L-proline I on dilution of a11 acetic or formic acid solution of form I1 with n-propaiiol or n-butanol. The revcrsihle nature of the form I form I1 transition seems to rule out a chcmical cahange, and suggests that the observed forward and rrversc mutarotations rcflect configurational transit ioris along iiidividual polymrr molerules. This view is supported by the finding that both form 1 arid form I1 of thc polymcr yicld L-proline on hydrolysis. Since poly-L-prolinr is cmmposrd elltirely of imino prptide linkages, the rhain is drvoid of any systematic. set of peptidr hydrogen bonds and one may question whethcr thc form I and form I1 structurcs repremit homogericous, ordcrcd configurations in solution. In considcring the various fac‘tors which cwuld act to stablize an ordrred configuration in solution, it may lj(1 srcii from IGg. 5 that the Ca-K bond of thc backbone chain is a cornponelit linkagc of t hc pyrrolidine ring and ronsrclucntly rotation ahout, thib hotid is impossible. The --j
~
0
//
C-N Bond
(1)
STRUCTURE OF COLLAGEN AND GELATIN
19
imide bond has a length of 1.34 A both in L-leucyl-L-prolylglycine and in the proposed structure for poly-L-proline I1 (Cowan and McGavin, 1955; Sasisrkharan, 1959a). Thus in the solid state this linkage behaves as a normal peptidr bond with an rnrrgy barrier to rotation rstimated a t about 21 kcal/molr (I'auling and Sherman, 1933). From an examination of spacefilling models it> is apparrnt that a drfinitr restriction to rotation also exists a t t hr
Bond (ii)
bond of t,he peptide backbone. Assuming t)he prpt,ide grouping to be in the trans-configuration, the neighboring pyrrolidine ring can assume two rotational posit,ions: (1) The carbonyl oxygrn is cis wit,h respect to thr hydrogen of the C,-at,om. I n this spatial arrangement (which has been termed cis'), rotation of the pyrrolidine ring about bond (ii) is limited to about 15". (2) The carbonyl oxygen is on the opposite side of the peptide bond with respect to the C,-hydrogen atom (trans'). This disposition allows the pyrrolidine ring a freedom of oscillation of about 60" about bond (ii). A similar set of restrictions is observed when the peptide grouping is cis. These rest,rictions severely limit the spectrum of configurational patterns available to a polymer of proline and, coupled with the fact that two distinct struct,ures are found in solution, indicated that these restraints play an important stabilizing role. However, it must not be assumed, given the planarity of the peptide grouping, that steric restrictions alone are sufficient for stabilization. Solvation phenomena appear to play a key role, as will be demonstrated below. When all of the peptide groups of poly-L-proline are in the trans-configuration and the groups about the (ii)linkage are trans', the resulting structure is the left-handed helix proposed by Cowan and McGavin for poly-Lproline I1 in the solid state. A photograph of a wire model built according to this arrangement is shown in Fig. 5. If all of the peptide bonds are disposed in the cis-configuration and t,he (ii) linkages are trans', a righthanded helix is generated (Fig. 5). In constructing an accurate model of this helix it, was observed that the helix dimensions conformed very closely to t,hose predicted by Cowan and Burge from the preliminary X-ray diffraction analysis of poly-L-proline I, ie., the helix has three residues per complete turn and a repeat distance of 6.30 A. It is also possible to construct, a right-handed helix with three and one-eighth residues per turn and a repeat of 5.85 A, as suggested by Crick and Rich, but in this case examination of wire models reveal that t8hepeptide bond grouping must be distorted significantly from coplanarity.
20
HAILRIKGTON .4ND VON HIPI'EL
Harrington m d Scla (1958) con(-luded from optical rotatory, sedinicnt ation, and viscosity studies that in thc form I 4 form I1 transition, a right-handcd hclix with cis-imide litikagcs was traiisformrd to a lcft-handed hrlix with trans-imide honds. The most pcrsuasivc argumcrit for this proposal conics from thc optical rotatory data which may bc uscd to calrulatc thc cotifigurational contrihution to thc spcrific rotation of mch hclix and thus to obtain an indication of helical seiis". Howevrr, hcfore such a cdculation c w i be made it is necebsary to estimatc thc residuc rotation of L-proline in a polypcptidc chain. This has been awomplishcd in thrw indcpcndciit ways (Harritigton and Scla, 1958; Striiiberg ct al., l!)(iOa). (1) T h c spwific*rotation of a serirs of glyc.inc-proline copolymers with incwasiug Gly/I'ro ratioh was mcasured. T h e specific rotation, corrcctcd for the wcight-fraction of prolinc, approachcd [a]i5= -2.50" at high Gly/I'ro ratios. ( 2 ) In aqurous solutions chontaining high conccntratiotis of JliBr or CaC12 , thc specific. rotation of poly-r,-proline I1 approachcs [a]'f = -2240". 'ITndcr thcsc (widitions the iiitririsic viscosity of the highly asymmctric form I1 strurturc dccreascs markcdly to valurs in the range of the globular protrins and it can hc assumcd that the configurational cwntribution to rotation of form I1 has becn climinated. ( 3 ) The rcsidur rotation of L-prolinr ('an also bc estimated from thr reported optical rotations of relativcly simple pcptidc dcrivativcs of proline and glyciiic. These mcthods yicld an averagr [a]i5 = -250" for the L-prolinc residur. Icrorn this valiic arid the spcrific rotations of form I ([a]:: = +10") arid form I1 ([a]i5= --51O0), the contribution of the right-handcd hclix to rotation is closc t o +300" whcrcas that of the left-handcd hclix is about - 290". (ii) The mechanism of mutarotation. Dircrt evidence for thc mrchanism responsible for mutarotation comcs from kinetic studics. Stcinbcrg rt al. (196Oa) ohscrved that the rates of both thc forward arid revws" mutarotatiori rcactions arr markrdly ac*ccleratcd by small amourits of strong arid. Thus 0.052 molc of ITC104 per mole pcptide bond rrduces thc half-life of mutarotation of form I -+ form I1 in acrtic acid from 5-15 to -1 min. Similarly addition of tracc amounts of H@1(I4(0.15 M ) accclcratcs the rate of revcrw mutarotation (form 11 -+ form I) in water-propanol (1:9 v/v) from a halftime of 444 min to 60 min. The small amount of acid required in these experiments suggests that the mechanism involves proton binding at the imide linkages of thr polymer. Other cvidencc supports this virw: when poly-L-proline is prcripitated from arctic arid solutions on addition of anhydrous pcrchloric acid, thc resulting prccipitatc contains, after thorough washing arid drying, about 0.30 molc tI(>l04 per molc peptide bond.
STRUCTURE OF COLLAGEN AND GELATIN
21
Potentiometric titration of simple proline derivatives and of poly-0-acetylhydroxyproline in acetic anhydride also demonstrate protonation of the imide linkages with about 0.35 mole proton bound per mole imide bond (Steinberg et al., 1960a). Protonation of simple amides has been found to markedly decrease the double bond character of the C-N amide linkage. Thus Berger, et al. (1959) found that protonation of the C-N bond in N-methylacetamide and N , N'-dimethylacetamide resulted in a depression of the double bond character as measured by nuclear magnetic resonance. In neutral aqueous solution the nuclear magnetic resonance spectrum shows the presence of two N-methyl lines indicating the absence of free rotation about the C-N linkage. In the presence of acid the doublet is replaced by a single absorption band with the onset of free rotation. Protonation appears to give an equilibrium between the three species:
(1)
(11)
(111)
of which free rotation is possible about the amide linkage in species (111). It is probable that a similar mechanism applies in polymers of proline, allowing cis trans-isomerizations at the peptide linkages. In the absence of steric effects the activation energy of the form I -+ form I1 transition should approximate the energy barrier to rotation about the peptide linkage (21 kcal/mole) provided that mutarotation involves the proposed cis + trans-isomerization. Downie and Randall (1959) measured the rates of forward mutarotation of poly-L-proline I in acetic acid at various temperatures and obtained an activation energy, AE* = 23 kcal/mole. The rate of the reaction was independent of concentration over a sevenfold dilution of the polymer. That is, a t any stage of mutarotation (as measured by [a]:) the velocity constant, lc, was found to be independent of concentration. On the other hand k decreased from 15 X sec-l to 2.5 X loF6sec -1 during the course of mutarotation. The kinetics of the optical rotatory changes of poly-L-proline in various solvents and at various temperatures have also been studied by Steinberg et al. (1960a). Inacetic acid the course of the forward mutarotation reaction was found to be independent of concentration (over the range 0.25 to 2.0 gm/100 ml) but, as observed by Downie and Randall, the rate constant depends on the degree of mutarotation. An activation enthalpy, AH* = 21 kcal/mole, was determined for both the forward mutarotation of form I in acetic acid and the reverse mutarotation of form I1 in acetic acid-n-propanol.
+
22
HARRINGTON AND VON HIPPEL
The type of mutarotation kinetics found experimentally is strongly dependent on the solvent. Forward mutarotation in acetic acid yields a plot of log d[a]/dt versus log ( [ a ] t- [a],J which is linear over 97% of the reaction with a slope of 1.33. On the other hand the rate is virtually invariant over two-thirds of the reaction in a solvent of 30% water-70% acetic acid. Reverse mutarotation in acetic acid-n-propanol appears to follow first-order kinetics over 90 % of the transformation. From these experiments and the fact that the enthalpies of form I and form I1 are essentially identical (Steinberg et al., 1960a) it seems likely that solvation is decisive not only in determining the kinetic pathway of mutarotation but’ also the structural pattern which is stabilized in a given solution. If we assume that mutarotation involves the rotation of randomly spaced proline residues about the backbone linkages, it can be seen that the rate of this isomerization need not remain constant throughout the reaction. Because of the geometry of the pyrrolidine ring, a cis 4 trans-isomerization at the peptide linkage virtually inverts the chain direction, leading to proximal interactions between neighboring rings and to steric interference to rotation of these vicinal residues. The average environment of any particular isomerizing residue may therefore depend on the degree of mutarotation. Steinberg et al. (1960a) have developed a kinetic analysis of this process assuming that each individual L-proline residue associated with a cis- or trans-peptide bond contributes to the observed specific rotation [a], according to the specific rotations of poly-L-proline I, [elI,and that of . poly-L-proline 11, [ a ] I IThus
where C c i aand Ctransare the concentrations of cis- and trans-peptide bonds, respectively, while Co = C c i s Ctrona,is the concentration of all peptide bonds. It follows that
+
-c e_i s -
co
[a1 - [a111 [a11 - b 1 I I
(2)
As pointed out above, the spontaneous cis trans-transformation requires proportionality between dC,i,/dt and Ccisa t any time. Hence,
but K is not a true reaction constant since we have seen that it varies throughout the course of mutarotation. The “constant” K is therefore a function of [a]or of the degree of conversion. The over-all mutarotation reaction may thus be represented by the equations
STRUCTURE OF COLLAGEN AND GELATIN
--dCcis - KCcis,
where K = f
at
23
(4)
or by
-dCci8 at
K’Ccis, where K’
=
dt)
(5)
The function K’ = &t) may be determined if the conversion can be expressed in terms of a reaction of a given order, p. In this case
where k(Cci,/Co)fl-lstands for K’(t) and k is independent of time and defines the initial rate of mutarotation. Integration within the time limits t = 0 to t (corresponding to Ccis = Co to CciJ yields (%>”-I
=
1
+
1 k(P
- l>t
(7)
or for the particular case under discussion
K’(t)
=
1
1
+ k ( P - 1)t
We have seen that the form I -+ form I1 interconversion may take place along different pathways depending on the solvent system, with differing over-all mutarotation kinetics. In acetic acid the apparent order of the forward mutarotation reaction is 1.33 (p). It will be seen from Eq. (8) that in this case K‘(t) should decrease with time, in agreement with the experimental observations. When p= 1, the mutarotation proceeds with firstorder kinetics. This behavior is seen in the reverse mutarotation of poly-Lproline I1 in acetic acid-propanol. When p < 1, K’(t) increases with time. This situation obtains in the initial stage of the forward mutarotation in acetic acid-water solvent which proceeds with zero-order kinetics. (iii) Hydrodynamic properties. The mutarotation of poly-L-proline I in propionic acid proceeds extremely slowly (A[a]z5 = -17” per 24 hr). Thus the molecular weight of a given polymer sample can be determined as form I and compared with that of the same sample after conversion to form 11. Combined sedimentation and viscosity studies on one sample of form I in propionic acid gave an estimated molecular weight of 19,000 After mutarotation to form I1 in acetic acid, the weight-average molecular weight was 18,400, while end-group analysis gave a number-average moleculrtr weight of 19,000. It thus appears that association or dissociation processes are not occurring during mutarotation in the simple aliphatic
24
HARRINGTON AND VON HIPPEL
acids, and that the fundamental unit particle in these solutions consists of a single polymer chain. The reduced viscosity (qsp/c) of form I1 is always greater than that of form I in a given solvent, consistent with the left-handed form I1 helix having a more asymmetric structure in solution than that of the form I, right-handed helix (see Fig. 5 ) . During mutarotation of form I 3 form I1 in acetic acid, the viscosity increases monotonically (Blout and Fasman, 1958; Downie and Randall, 1959; Steinberg et al., 1960a; Steinberg et al., 1960b). However Harrington and Sela (1958) observed marked reduction in I 1.2
-
6
\
g
I
I
I
I
I
I
-
-
1.0
0.8
v
2 0.6 0.4
0.2
0
I 0
I
I
100
200
A !
300
I 400
I I 500 600
-[a]~"' FIQ.6. Reduced viscosity (C = 1%) of poly-L-proline at 30°C as a function of [a]:. 0-0, datatakenduring forward mutarotationinglacial acetic acid; A-A, data
taken during forward mutarotation in acetic acid-water (7:3 v/v); 0-0,data taken during reverse mutarotation in acetic acid-n-propanol (1:9 v/v); A, value in aqueous 12 M LiBr. (From Steinberg et al., 1960b. Reproduced with kind permission of the American Chemical Society.)
viscosity during the early phase of the mutarotation in water as solvent. In these experiments the viscosity passed through a minimum, increasing during the later stages of the reaction. Steinberg et al. (1960a, b) reported a similar depression in the viscosity versus time plots in studies of the form I + form I1 interconversion in a solvent of 30 % water-70 % acetic acid. It is apparent that the order of the reaction [4/3in acetic acid, zero (initially) in water and in water-acetic acid] is related to the pathway of interconversion which is reflected in the hydrodynamic properties. Figure 6 illustrates this situation graphically. In various solvent systems the poly-Lproline molecules may assume different average configurations as measured by viscosity during mutarotation but still exhibit the same optical rotation. I t may be questioned whether the restrictions to rotation about the
STRUCTURE OF COLLAGEN AND GELATIN
25
backbone discussed above are sufficient to lead to rodlike particles for the form I and form I1 structures in solution. Fair agreement is found between the axial ratio estimated from viscosity studies (assuming a rigid prolate ellipsoid) and that expected from the coordinates of the left-handed Cowan-McGavin helix for low molecular weight polymers (Harrington and Sela, 1958). At molecular weights of 12,000 and above the axial ratio found is consistently lower than that expected, this deviation increasing with increasing molecular weight (Steinberg et aZ., 1960a). A similar examination of form I polymers for two different molecular weight samples (12,000 and 19,000) gave axial ratios of 29 and 37. Assuming that the form I chain in solution is a right-handed helix with all peptide bonds in the cis-configuration, the axial ratios calculated using the Rich and Crick (1958) dimensions are 27 and 29, respectively, whereas 31 and 45 were obtained using the Cowan and Burge (1958) dimensions (see Fig. 5). Flexibility in the form I chain becomes apparent at higher molecular weight. A sample of molecular weight 52,000 (Blout and Fasman, 19.58) gave an axial ratio from viscosity measurements of 44 whereas the expectled value is 116 (Rich and Crick) or 137 (Cowan and Burge). (iiii) The efect of neutral salts on the poly-L-proline 11 configuration. As mentioned earlier, the optical rotation and viscosity of poly-L-proline I1 are strikingly altered in the presence of certain neutral salts. The specific rotation of poly-L-proline I (DP = 200) approaches [ag5 = -240' when form I1 polymer is transferred from water to a concentrated aqueous lithium bromide solution, while the intrinsic viscosity falls from [ q ] = 0.54 dl/gm to [7] = 0.07 dl/gm. Similarly, in the presence of 4 M sodium thiocyanate the specific rotation of poly-L-proline I1 (DP = 550) is depressed to [a]E5= -290" while [q] decreased from 0.67 (in water) to 0.07 in this salt medium. The most likely explanation of these changes is that they result from destruction of an asymmetric, homogeneous structure (Harrington and Sela, 1958). In this connection it is of interest that a sample of PO~Y-DLproline (DP = 100) exhibited an intrinsic viscosity in water of only 0.05 dl/gm, indicating a lack of configurational asymmetry. This value remained unchanged in the presence of lithium bromide (Steinberg el al., 1960a). Although destruction of the structural integrity of the poly-L-proline I1 helix in the presence of lithium bromide, calcium chloride (Harrington and Sela, 1958), and sodium thiocyanate (Blout and Fasman, 1958) must involve rotation of the pyrrolidine rings about the backbone chain, present evidence indicates that loss of rigidity occurs at the
ce-c
//
0
Bond (ii)
26
HARRINGTON AND VON HIPPEL
linkages rather than a t the peptide bonds. For one thing, the N-methyl doublet observed in the nuclear magnetic resonance spectrum of N , N’dimethylacetamide remains unchanged when this substance is transferred from water to concent)rated aqueous solutions of lithium bromide or sodium thiocyanate (Steinberg et al., 1960a). Moreover, on massive dilution of a concentrated aqueous lithium bromide solution of poly-L-proline I1 with water a t low temperature (3°C-1 l°C), the resulting solution undergoes an extremely rapid mutarotation, with a rate about lo3 faster than that expected for the “normal,” acid-catalyzed reaction. The activation enthalpy of this reaction (AH* = 21 kcal/mole) is about the same a s that calculated for the acid catalyzed mutarotation, but the entropy of activation is markedly different (AS* = 0.84 e.u. for the dilut,ion reaction and -12.5 e.u. for the acid-catalyzed reaction) demonstrating that fundamentally different mechanisms are involved in the two processes. It seems probable, therefore, that configurational changes in the three-dimensional architecture induced by neutral salts result from rotations of the rings about the (ii) linkages. Since the helical pattern of form I1 is eliminated in the presence of neutral salts, steric restrictions between contiguous groups of the chain cannot be the primary source of stabilization a t bonds (ii). It would appear more likely that the role of the neutral salts is to modify the helix-solvent interaction. 3. Poly-L-hydroxyproline
Poly-L-hydroxyproline was first synthesized (Katchalski et al., 19,iCi; Kurtz et al., 1958a,b) through polymerization of O-acetyl-N-carboxyhydroxy-L-proline anhydride in pyridine followed by deacetylation in aqueous ammonia. The polymer is quite water-soluble, insoluble in the simple aliphatic acids, but does not exhibit the mutarotation properties of poly-Lproline I, probably because of the prolonged treatment in alkaline solution required for deacetylation. Aqueous solutions of poly-L-hydroxyproline exhibit an = -400” which is invariant with time. The magnitude of the specific levorotation suggests that the backbone chain of poly-L-hydroxyproline is arranged in a structural pattern in solution similar to that of poly-L-proline 11. This proposal is supported by the optical rotatory behavior of the polymer in aqueous lithium bromide. Addition of the salt lowers the specific rotation to [a]i6 = -168”, paralleling the drop in levorotation observed in poly-L-proline I1 under these conditions. The optical rotatory dispersion constant, A,, falls from 206 to 191 mp, which is also comparable to that observed for poly-L-proline I1 (A, = 202 mp in water; X, = 185 mp in 8 M LiBr). Sasisekharan (195Yb) has recently proposed a structure for poly-L-hy-
STRUCTURE OF COLLAGEN AND GELATIN
27
droxyproline in the solid state on the basis of X-ray diffraction patterns obtained from powders and oriented films. The polymer chains appear t o be packed in a hexagonal lattice. Each polypeptide chain takes up essentially the same left-handed configurational pattern as that of poly-L-proline 11, but the fundamental unit cell requires three helices as in collagen. Although mutarotation of poly-L-hydroxyproline has not been observed because of the conditions used in the deacetylation step, the acetylated derivative, poly-0-acetyl-L-hydroxyproline, undergoes marked optical rotatory changes with time in solution (Kurtz et al., 1958a, b). Immediately after dissolution in formic acid, this polymer shows a specific rotation, [a]:5 = +25" (form I). On standing 6 hr a t room temperature the solution becomes strongly levorotatory with [a]:5 = -175" (form 11). Reversal of the mutarotation (form I1 -+ form I) could be effected by boiling in N,N'-dimethylformamide, by diluting a solution of form I1 in formic acid with 40 volumes of pyridine or by dissolution in acetic anhydride (Steinberg et al., 1960a). Addition of trace amounts of a strong acid (HClOh) accelerates both the forward and reverse mutarotation of poly-O-acetyl-L-hydroxyproline indicating, as in the case of poly-L-proline, that isomerization is taking place at the peptide linkages. If relatively large amounts of perchloric acid are used, the specific rotation of the polymer immediately changes to values intermediatebetween those of form I and form 11, being uniquely determined by the ratio of acid/peptide bond. Since the same final specific rotation is achieved on addition of a fixed amount of perchloric acid to represent true form I or form 11, it appears that the various levels of equilibrium states and that these reflect an equilibrium between the cisand trans-peptide group configurations in each molecule, which are in turn determined by the extent of protonation. From titration studies it appears that the polymer exists in the form I1 configuration after protonation of about one-third of the peptide linkages. This situation is similar to that found on titration of poly-L-proline. Downie and Randall (1961) have recently measured the mutarotation of poly-0-acetyl-L-hydroxyprolinein formic acid and the reverse mutarotation in N ,N'-dimethylformamide at various temperatures and polymer concentrations. The activation energy of forward mutarotation was 22.4 kcal/mole and of reverse mutarotation, 23.8 kcal/mole, consistent with a trans- cis-isomerization mechanism involving rotation about the peptide bonds. Mutarotation phenomena are also exhibited by another derivative of poly -L-hydroxyproline, i.e., poly-0-p-tolylsulphonylhydroxylL-proline (Kurtz et al., 1957, 1958a). On dissolution in acetic acid, this polymer gives
28
HARRINQTON AND VON HIPPEL
[a]:' = 0" (form I) which changes to a terminal value, [a]:' = -120", on standing (form 11). Dilution of the acetic acid solvent with pyridine (40:1 v/v) brings about a reversal in mutarotation.
4. Copolymers Containing L-PTO~~TM
Copolymers of L-proline and glycine have been prepared by polymerizing mixtures of N-carboxy anhydrides of proline and glycine in various weight ratios. Copolymers were obtained with molecular weights ranging from 950 to 2050 as judged by end-group titration, and in mole ratios proline: glycine (PG,) from PGa.a to PGs (Kurtz et at?., 1958s; Steinberg et al., 1960a). Only the copolymers with low glycine content (below PG2) were TABLEI F Optical Rotation of Copolymers of L-Proline and Ulycine Polymer
Poly-L-prolineI1
PzG PG
PG2
PG: PGs PGa 0
Number Molar ratio average of residues molecular weightb
1:0.66 1:1.06 1:2.2 1:3.3 1:4.9 123.8
14,300 950 2000 1700 1400 1630 2060
Solvent
blDc
Formic acid Formic acid Trifluoroacetic acid Trifluoroacetic acid Trifluoroacetic acid Trifluoroacetic acid Trifluoroacetic acid
-640"
-540"
-316" -271" -190" -136" -90" -41"
-438" -440" -435" -398" -348" -256"
[dD.oorreotadd
From Kurtz et al. (1960).
b
End group.
d
Corrected for index of refraction of solvent. Based on weight-fraction of proline.
found to be water-soluble. All of the copolymers were quite soluble in trifluoroacetic acid and in concentrated aqueous lithium bromide solutions, but only slightly soluble in glacial acetic acid. Mutarotation of a copolymer with a molar ratio of 1:1 (PG) has been observed very recently by Downie and Randall (1961). In glacial acetic acid the initial specific rotation ([a]:') was -118" which changed to -257" on standing at 25°C. Similar behavior was observed in aqueous solution. The effect of glycine residues in diluting out the poly-L-proline II-type configuration may be seen in Table 11, which presents the specific rotation of a series of L-proline-glycine copolymers in trifluoroacetic acid. As the glycine content increases, bhe helical contribution of the left-handed form I1 structure is eliminated (last column) and a t the highest g1ycine:proline mole ratios (PGs) the specific rotation approximates that of the proline
29
STRUCTURE OF COLLAGEN AND GELATIN
residue. The disappearance of the form I1 structure with increasing glycine content is also apparent in Fig. 7,where the specific rotation of each of the copolymers is measured in varying concentrations of lithium bromide. The effect of this salt on specific rotation becomes progressively less as the
0 DP-50
OP.30
t
-0
1:9
-0-0
1
OO
4
8
12
L i B r CONCENTRATION ( M )
.
FIQ.7. Specific rotation of various L-proline-glycine copolymers as a function of lithium bromide concentration. Insert, la]:’ versus LiBr concentration for two samples of poly-L-proline 11. (From Kurtz et al., 1960.)
number of contiguous proline residues are decreased, suggesting that neighboring pr pro line residues are required to give the observed salt effect. This conclusion is entirely consistent with the studies discussed above on poly-L-proline 11. These experiments lead us to consider another aspect of the configurational properties of proline-containing polypeptide chains which can be stated in the form of a question: How many contiguous proline residues
30
HARRINGTON AND VON HIPPEL
are required to achieve the complete helical contribution to optical rot,ation characteristic of poly-cproline? To answer this question Yaron and Herger (1961) have recently prepared a skeleton copolymer consisting of an optically inactive poly-DL-lysine backbone bearing short polybenzyl-maspartate side chains. The side chains were used as sites for attachment of poly-L-proline segments of varying length. It was found that the specific rotation of such copolymers undergoes a sharp change to values characteristic of high molecular weight poly-L-proline at chain lengths between 4 and 6 proline residues. In this range an abrupt transition was also observed in the kinetics of mutarotation of the copolymers. I n formic acid the specific rotation (based on the weight fraction of proline) approached the value expected for form I1 whereas examination before mutarotation showed a similar transition to the specific rotation characteristic of form I. Copolymers of L-proline and sarcosine have been prepared by Fasman and Blout (1961). These were found to exist in two forms, analogous to poly-L-proline. Form I, which is obtained directly from the copolymerization mixture showed anomalous optical rotatory dispersion and relatively low viscosity. On dissolving form I in 2-chloroethanol, form I1 is obtained which exhibits normal optical rotatory dispersion and relatively high viscosity. Fasman and Blout have suggested that the transition, form I -+ form 11, involves a conversion of the structure from cis- amide bonds to trans-amide bonds.
111. THE COMPOSITION AND AMINOACID SEQUENCE OF COLLAGEN AND GELATIN A. Amino Acid Composition I n recent years the use of ion-exchange chromatography has greatly improved the accuracy and speed of amino acid analysis, and in the case of collagen and gelatin has provided us with complete compositions from nearly every class of vertebrates. A number of invertebrate collagens have also been analyzed. Chemically, collagen is characterized by an unusually high content of glycine and the imino acids proline and hydroxyproline; the presence of hydroxylysine; and notably small amounts of aromatic and sulfur-containing amino acids. The unusual fine structure of the polypeptide chains of collagen, mirrored in the wide-angle X-ray diffraction patterns, and the characteristic gross structural features, which are recognized by smallangle X-ray diffraction and electron optics, are both intimately related to this unusual composition. Our purpose in the present section is to draw attention to those aspects of the chemical studies which may help in throwing light on the fine structure of the protein. It is clear that any detailed proposal of the molecular architecture of collagen must be compatible with
STRUCTURE OF COLLAGEN AND GELATIN
31
the composition of a wide range of collagen species. Moreover, a n examinat,ion of the compositions should help to characterize the chemical features which link members of the collagen class of proteins.
1. Vertebrate Collagens Much of the recent work on the amino acid composition of this class has been collected by Eastoe and Leach (1958). Table I11 summarizes data from the studies of Neuman (1949), Tristram (1953), Eastoe (1955, 1957), Leach (1957), and Piez and Gross (1960) and includes both collagens and gelatins. From a careful comparison of the compositions of collagen and their derived gelatins, Eastoe (1955) and Eastoe and Leach (1958) have given convincing evidence that the amino acid composition of collagen is faithfully reproduced in the derived gelatin. I n fact from the chemical point of view, Eastoe and Leach (1958) consider the preparation of gelatin to be a purification of the parent collagen. An examination of Table I11 reveals that the amino acid compositions of mammalian collagens are very similar over a wide spectrum of species. Very close to one-third of the total residues are glycine, about one residue in ten is hydroxyproline, and twelve in a hundred are proline. Tyrosine, histidine, and the sulfur-containing amino acids are present at concentrations of less than 1 %. Eastoe and Leach (1958) suggest that the tyrosine associated with the parent collagen may be, in part, an impurity since the concentration of this amino acid is reduced in the derived gelatins, and further decreased when these are fractionated with ethanol. Although hydroxylysine is also present a t very low concentration (3 to 12 residues/ 1000) it cannot be removed by purification procedures and is now thought to be, along with hydroxyproline, a requirement for admission of a protein to the collagen class. These two hydroxyl residues seem to be unique to collagen among animal proteins. It is of interest that the protein of the nemocysts of Hydra and Physalia appears to contain as much as 20% hydroxyproline (Lenhoff et al., 1957). Hydroxylysine has been detected in the free state in embryonic muscle extracts (Gordon, 1948). The fish collagens exhibit an appreciably wider range of composition than the mammalian species, in keeping with the greater evolutionary time scale which they span. For example, the Australian lungfish, which is one of the four surviving species of the class Crossopterygii is thought to be more closely related to land vertebrates than any living fish (Young, 1950). It is therefore of considerable interest that the amino acid composition of this species approaches that observed for the mammalian collagens. Similarly, the cod (Actinopterygii) which is among the most recently developed of the bony fish, exhibits the greatest divergence from the typical mammalian composition (Eastoe, 1957).
TABLE
111
Amino A r i d Composition of Vertebrate Collagens and Gelatins0 I
l
l
1
ox -
Amino acid
skin (C) -
Alanine Glycine Valine Leucine Isoleucine Proline Phenylalanine Tyrosine Serine Threonine Methionine Ar gin i n e Histidine
Lysine Aspartic acid Glutamic acid Hydroxyproline Rydroxylysine Amide groups
-._pI
_ _ ~ _ _ _ _
99.6 12 109.7110.8 99.9106 110.7 110.5 114.6 138 i20 314 326 327 327 324 326 331 21.2 21.9 25.1 22 25.4 20.6 19.8 27.1 20 39.9 25 27.9 23.7 25.4 25 26.0 24.8 23.8 - 11 12.3 9.6 12.7 10 11.1 11.0 10.9 122.3 38 118.8 130.4 120.0 117 126.4 128.2 129.5 14.1 13 16.3 14.4 13.6 13 14.2 13.0 14.3 5.1 2.6 2.9 3.2 4 . 8 3.2 3.6 3.6 3.4 29.9 36 37.8 36.5 27.9 41 36.9 41.0 28.6 17.9 18 19.7 17.1 20.6 20 18.5 24.0 19.1 5.0 4.3 5.1 5.4 5.7 6.3 5.7 4.7 6.2 46.0 50 49.0 48.2 49.9 49 49.0 50.1 44.8 4.5 5.0 5.8 6.0 4.2 5.1 5.4 5.7 4.5 26.2 26.2 35.5 29 21.6 25.9 19.0 28.6 27 48.4 46.3 48.1 44.0 45 49.8 46.8 49.2 47 71.7 72 75.8 72.0 76.4 74 72.3 69.6 74.1 92.1 89.1 98.5 99.6 94 100.8 95.5102.4100 6.3 7.4 6.4 5.9 5.7 8.9 5.8 9.6 41.8 40.8 - 51 44.0 25.6 40.1 43.9 46
-__--__
18.6 91.3 -
Referencec
(1)
(6)
18.3 18.3 17.9 92.4 91.3 92.4 -
17.9 18.6 17.8 91.6 91.1 91.1
(2)
(2)
(2)
r.G
jwlm Carp- Cod- Pikeblad- skin skin skin der ( G ) ( C ) (GI (C)
I
Total N Mean residue weight
w
C?T
(3)
(7)
a Residues of amino acids per lo00 total residues. ~References:(1) Tristram (1953). (2) Eastoe (1955). (7) Gross and Pie2 (1960) [6) Piee and Gross (1960).
(2)
(4)
114.0 125.0 98.0'128.0 119.0 324 315 301 311 333 15.4 20.2 21.9 21.3 21.9 20.1 25.7 28.8 25.2 23.9 11.4 11.7 14.0 12.2 19.4 127.9 119.4 109.7 126.0 113.4 17.7 14.2 19.3 15.3 13.9 3.3 1.8 6.1 1.1 1.4 42.1 43.6 66.3 43.7 44.5 22.0 17.9 26.4 26.1 25.8 6.5 6.1 8.7 4.0 10.0 49.5 49.9 49.2 51.0 50.3 4.7 4.7 6.5 5.1 7.4 25.3 27.6 29.1 24.2 24.3 45.5 48.0 54.9 48.6 42.6 72.8 62.4 77.9 78.9 65.8 92.8102.0 77.5 73.1 78.5 4.9 4.0 4.3 5.3 4.7 25.5 22.1 53.9 46.8 29.4
118.9 337 18.0 17.7 11.4 102.2 14.1 2.4 50.5 29.2 8.8 52.4 4.8 21.8 47.5 70.5 82.0 10.7 41.0
18.3 16.2 18.0 18.2 18.2 91.4 91.2 93.3 91.4 90.8
18.5 90.7
(4)
(5)
* Abbreviations:
(4)
(4)
(5)
(5)
126 120 107 114 325 317 345 328 18 19 19 18 21 23 20 25 9.2 10 12 11 116 124 102 129 14 14 13 14 2.0 3.2 3.5 1.8 37 43 69 41 29 27 25 25 13 12 13 12 53 53 51 45 3.8 4.5 7.5 7.4 26 27 25 22 47 52 47 54 71 74 75 81 81 73 53 70 7.4 4.5 6.0 7.9 38 26 33 42
~
~
-
_
_
-
_
_
-
C = collagen; G = gelatin 3 = extract. (5) Eastoe (1957). (3) S e u m a n (1949). (4) Leach (1957).
E 5
$
4
$
g
P
_ I
STRUCTURE OF COLLAGEN AND GELATIF;
33
In agreement with earlier work (Gustavson, 1955a), the total imino acid content within the fish group is significantly lower than that of the mammalian collagens, while the hydroxyamino acids serine (Neuman, 1949) and threonine (Beveridge and Lucas, 1944) and sometimes hydroxylysine are enhanced, leaving the level of hydroxyl groups about the same in both the fish and mammalian series. The methionine content of fish collagens is increased over that of the mammalian collagens except in the lungfish, while tyrosine and histidine remain at less than 1 % of the total amino acids throughout the series. Although significant variations in composition are found among the fish collagens, the glycine content remains essentially invariant throughout all species a t close to one-third of the total amino acid residues (Piez and Gross, 1960). Since the early work of Grassmann and Schleich (1935) and Beek (1941), it has been known that a large variety of sugars are associated with the collagens and gelatins. In addition to the small amounts ( < 1 %) of glucose, galactose, and mannose reported by Grassmann and Schleich, glucosamine (Schneider, 1940), fucose (Glegg et al., 1953), ribose, arabinose, and galactosamine (Gross et al., 1958) have been found. The amounts of carbohydrate determined vary, depending on the species and the degree of purification of the collagen, but generally add up to less than 2 % by weight. Purification or gelatinization, followed by fractionation, usually reduces the carbohydrate content significantly. On repeated precipitation of soluble collagens, the glucosamine disappears and the hexose content drops to about 0.7% (Wolf, 1956; Grassmann et al., 1957b). This concentration can be further reduced through oxidation with sodium periodate, and after a short oxidation step only 0.15-0.20% of the hexoses remains (Hormann and Fries, 1958; Kuhn et al., 1959). Since this residue cannot be destroyed by oxidation, Schneider (1940) and Grassmann el al., (1957a) have suggested that it, may be chemically bound to collagen through 0-glycosidic linkages. 2. Invertebrate Collagens
At the present time complete amino acid analyses have been reported on nine of the invertebrate collagens: the cuticle of the segmented roundworm Lumbricus (Singleton, 1957; Watson and Silvester, 1959) and nonsegmented roundworm Ascaris lumbricoides (Watson, 1958), ejected Cuverian filaments of Thyone (Watson and Silvester, 1959), two spongins from Sp. graminea, the collagen of the float of Physalia, the body walls of Metridium and Thyone (Piez and Gross, 1959), and body wall of the garden snail Helix aspersa (Melnick, 1958; Williams, 1960). Table IV gives the quantitative amino acid analyses reported for these species. Possibly the most striking feature of Table IV is the contrast between
TABLE IV Amino Acid Composition of Invertebrate Collagens and Gelatinso Echinoderm Amino acid
Alanine Glycine Valine Leucine Iso1eu cine Proline Phenylalanine Tyrosine Serine Threonine Methionine Arginine Histidine Lysine Aspartic acid Glutamic acid Hydroxyproline Hydroxylysine Cystine Amide groups Glucosamine Galactosamine
Body wall (G)b
:' (H jwskdi) duverian
cw) Cuticle
fibers
(GI
113 306 30 22 13 109 8.9 7.9 43 35 2.2 54 2.8 7.5 62 110 60 11.o 2.5 75
114 308 21 28 13
84
21 0 15 56 81 165 0 97
2.4 0
-
-
81 11 11 55 51 6.9 46 4.8 12 70 77 54 4.7
-
e3 !-P
Coelente-
103 324 17 29 15 13 5.2 0 105 52 0
-
Mollusca
(Ascaris)
km) Body wall (GI
Float (G)
Spongin A
'POngin
69 286 13 18 14 280 7.3 1.2 23 18 12 42 8.4 37 76 101 24
70 311 34 37 23 63 12 7.9
44
56 315 29 28 24 78 9.3 4.7 38 43 4.7 47 3.9 9.0 92 95 108 12.0 3.3 102
94 323
94 49 25 3.2 71
66 307 26 31 22 83 11 5.6 47 33 5.8 54 1.9 27 83 104 61 30 1.6 66
-
4.0 0
2.5 3.2
10 7.7
(G)
0
-
54
39 8.8 57 5.1 27
80
0 R.asidues of amino acids per lo00 total residues. * Abbreviations: G = gelatin. (3) Watson (1958). (4) Williams (1960). (2) Watson and Silvester (1959).
c
24 24
17 73 10 4.0 24
27 3.1 43 3.2 24 97 86 94 24 6.0 90
1.6 0
Body (G)
72.3 321 21.5 23.5 12.1 104.1 9.9 8.8 61.4 27.7 1.2 50.9 2.6 8.3 66.8 99.1 99.5 8.2 0.0 46 .92 2.5
References: (1) Pies and Gross (1959).
P
3 4
0
2
STRUCTURE OF COLLAGEN A N D GELATIN
35
the remarkable invariance of the glycine content and the wide range in composition seen for most of the other amino acids. As in the vertebrate collagens, glycine makes up nearly one-third of all of the amino acids and it seems clear that this level of glycine must have a fundamental relation to the collagen structures. The total imino acid content varies from 112 to 304 residues per 1000, but a much wider variation is observed in the individual imino acids, with proline varying from 13 to 280 residues per 1000 and hydroxyproline from 24 to 1G5. Gelatins from Physalia float, Metridium body wall, and spongin B have a high content of hydroxylysine, whereas earthworm and roundworm cuticle apparently are devoid of this amino acid and the former collagen is also lacking in histidine, tyrosine, and methionine. The invertebrate collagens generally have a larger proportion of polar amino acids, smaller amounts of imino acids, and more total hydroxyamino acids than do the vertebrates, while the aromatic and sulfurcontaining residues are consistently low. A large variety of sugars are also found associated with the invertebrate collagens. Chromatographic analyses reveal the presence of glucose, galactose, glucosamine, galactosamine, fucose, mannose, and in some cases arabinose (Gross et al., 1958). In contrast to the vertebrate connective tissues, purification and gelatinization appears to be much less effective in removing polysaccharide material. Furthermore, the sugar content is generally much higher than in the vertebrates, ranging from about 18 % in spongin A to 5 % in Thyone corium (Gross and Piez, 1960). At the moment there is insufficient evidence to permit a decision as to whether the sugars form an integral part of the invertebrate collagens or are simply associated with them physically. All of the invertebrate collagens which have been examined have yielded t,he typical collagen wide-angle X-ray diffraction pattern, and electron micrographs of the fibers show, with the exception of Physalia float and Metridium body wall, the characteristic 600-700 A axial period (Gross et al., 1958).
B. Amino Acid Sequence I. Peptides Derived from Acid and Basic Hydrolyzates
In recent years, the isolation and identification of a large number of di-, tri-, and tetrapeptides from acid and basic hydrolyzates of collagen and gelatin have been achieved. Numerous longer peptides have also been separated from tryptic and collagenolytic digests, and the composition, and in some cases the partial sequence of these peptides have been determined. We now have at hand enough information from these studies to establish the broad outlines of the primary structural pattern predominating in the polypeptide chains of collagen.
30
HARRINGTON AND VON HIPPEL
In Table V are collected the most important peptides isolated by Heyns et al. (1951), Schroeder et al. (1953), 1954), and Kroner et al. (1953, 1955), following acid hydrolysis of steer hide collagen and gelatin. The peptide fractions were separated on ion exchange columns, and individual peptides resolved on celite, followed by sequence studies carried out by the methods of Sanger. The widespread distribution of glycine among the different peptides of Table V, coupled with the fact that this amino acid makes up one-third of the total residues of collagen, suggests that glycine occurs at every third residue. It is apparent that this arrangement is not absolute, however, since some peptides with the sequence Gly.Gly have been identified. The presence of contiguous, identical residues such as Gly.Gly and Ala.Ala and the variation in the type of amino acid residue associated with the amino group of glycine, rule out extensive regular repeating patterns such as -Pro.Gly.X.Pro.Gly.X- suggested by Astbury (1940). This structure, as well as one made up of the repeating sequence -Gly.X.Pro.Gly.X.Xproposed by Bergmann (1935) and Bergmann and Niemann (1938) cannot be correct in view of the wide variation in total imino acid content encountered in the vertebrate (fish) and particularly in the invertebrate collagens (see Tables I11 and IV) . Nevertheless, examination of Table V reveals that the imino acids seem to be distributed through the isolated peptides in a remarkably systematic manner. Thus, almost all of the imino acids occur next to glycine, either as Gly.Pro or as Hypro.Gly. Schroeder et al. (1954) have suggested that the isolation of such a large quantity of Gly.Pro indicates a very marked lability toward acid hydrolysis of the peptide bond associated with the carboxyl group of proline, while the high level of Hypro.Gly is evidence of the lability of the peptide bond associated with the imino group of hydroxyproline. Since proline and hydroxyproline are present in about equal amounts in steer hide gelatin, it was inferred that sequences of the type -Gly.Pro.Hypro.Gly- could be an important pattern in the primary structure. The work of Kroner et al. (1955) has supported this proposal with the identification of the tripep tide Gly.Pro.Hypro and isolation of the tetrapeptide Gly.(Hypro, Pro) .Gly from partial acid hydrolyaates of steer hide collagen. From the discussion given in Section 11, it is apparent that the presence of neighboring imino acid residues has a profound effect on the chain geometry in these regions. Since insertion of a single proline results in rotation of the chain by -120", two contiguous residues would give the chain a left-handed twist of -240" and, assuming the usual restriction to rotation of neighboring pyrrolidine rings, would generate an incomplete element of the poly-L-proline I1 helix along the chain. This feature may be of fundamental significance to the structure and will be considered in more detail in later sections.
TABLE V. Peptides from Acid and Basic Hydrolyzatea of Collagen and Gelatin' Neutral peptides
Basic peptides
Peptides with add and basic amino acids
Acidic peptides __
Ala.Ala Ala.Gly Gly.Ala Val.Gly Ser.Gly Ser.Ala Thr.Gly Thr.Ala Gly.Gly Leu.Ala Ala.Gly.Ala Ala.Ala.Gly
4.6~ 13.0. 9.oc 4.lc 18.40 1.5O 17.40 1.1"
Ala.Arg Ala. (Arg,Gly) Ala.Lys Arg.Gly .Gly Lys.Gly Ser.Arg
Gly.Asp Gly .Glu Ala.Asp Val.Glu Leu.Glu Glu.Gly Glu.Ala Glu .Asp.Gly
1.oc
Asp.Arg 7.0. ASP.(Arg,GW 1.9c Glu.Arg 0.5~ Gly .Arg.Gly 0.4~ 4.5c 6.6. 0.5d
d -d
12.4
-d
--. __
69.1
-
1.20 Gly.Pro 0.7~Gly .Pro .Gly 1 . 8 c Gly .Pro .Ala 1 . o c Gly .Pro .Glu - Ser. Pro.Gly 4.7 Ala.Pro .Gly Lys.Pro.Gly Pro. (Gly,Lys) Pro.Ser Pro.Thr Hypro.Gly Ala.Hypro.Gly Ala.Hypro Leu.Hypro Ser.Hypro.Gly Glu.Hypro Glu.Hypro.Gly Gly.Pro.Hypx-o Gly. (Hypr0,Pro.o).Gly
I
Taken from Grassmann (1955). b In micromoles peptides per 250 mg of gelatin or collagen. 0 Peptides have been assembled from the work of Schroeder et al. (1953,1954). d Peptides have been assembled from the work of Kroner et al. (1953,1955). Peptides have been assembled from the work of Heyns et a2. (1951). a
Peptides containing proline and hy droxyproline
-
61.80 0.4d 3.5" 0.7d 0.5" 0.3d A
1.0= 1.oe 0.7~
35.6. 3.6d 1 .Od 1.7~ 1 .4d 0.9d 2.4d 4.2d 3.ld
23.8
-
38
HARRINGTON AND VON HIPPEL
2. Peptides Derived from Enzymatic Hydrolyzates a. Tryptic Digests. The acid and basic hydrolyzates of collagen and gelatin have given much valuable information, but it is clear that the limited size of the isolated peptides prevents a detailed elaboration of the primary structure. What is obviously needed is sequence information on much longer peptide segments and it may be expected that the use of specific enzymes, a technique employed so successfully in the delineation of the primary structures of insulin and ribonuclease, will again prove indispensable. Although the task is a formidable one, Grassmann, Hannig, and their co-workers (Grassmann et al., 1956, 1960; Grassmann, 1960) have made substantial progress in the separation and characterization of tryptic hydrolyzates of calfskin collagen. Following digestion of a large mass of purified, heat-denatured soluble collagen (62 gm) with chymotrypsin-free trypsin, 51 homogeneous peptides with lengths varying between 3 and 131 residues were isolated through a combination of preparative “continuous” curtain electrophoresis and column arid paper chromatography. The 51 peptides represent about 55% of the total initial protein. N-terminal and C-terminal end groups have been determined for 18 of the peptides, establishing glycine in the N-terminal position for every peptide and either lysine or arginine in the C-terminal position. Of the 51 peptides, 90% contain between 30 and 38% glycine, giving very strong support to the contention that, with a few exceptions, this amino acid appears at every third residue throughout the component chains of collagen. A few peptides have been analyzed in which the glycine content was significantly higher than this and partial sequence work revealed two glycine residues neighboring each other or separated by a single amino acid (Grassmann, 1960). On the other hand, three peptides were isolated which were devoid of glycine over 4 t o 5 residues. Proline- and hydroxyproline-rich peptides were found, in general, to be deficient in diamirio and dicarboxylic acids, while peptides deficient in imino acids contain large quantities of polar residues. Following the early suggestions of Bear (1952), Grassmann and his collaborators (Kuhn el al., 1958a, b ; Grassmann et al., 1960) have particularly emphasized the possibility that the periodic occurrence of these regions could be responsible for the characteristic band structure observed in the electron-optical investigation of phosphotungstic acid-stained collagen fibrils (Hall et al., 1942; Schmitt and Gross, 1948), with the apolar, imino-rich regions corresponding to the light “interband” segments and the polar, imino-poor regions to the dark “bands.” In this connection, investigation of a peptide consisting of 43 or 44 amino acids (Grassmann et al., 1956) revealed that the six N-terminal residues contained no imino acids, a central fragment
STRUCTURE OF COLLAGEN AND GELATIN
39
of 33 amino acids was made up primarily of proline, hydroxyproline, and glycine (10 Pro, 5 Hypro, 12 Gly, 2 Ala, 1-2 Ser, 2 Tyr, 1 Glu) while the 5 C-terminal amino acids included 3 dicarboxylic acids, but no imino residues. Similarly, two separate peptides containing 22 and 21 residues were completely devoid of either proline or hydroxyproline, but yielded 41 and 33 mole % polar amino acids, respectively. When the minimum molecular weight of a number of the isolated peptides estimated from quantitative end-group determination is compared to that expected from amino acid analysis, a surprising result emerges (see Table VI). Twelve of the 18 peptides examined exhibit minimum molecular weights by amino acid analysis which are, within experimental error, three times that expected from the N-terminal analysis, suggesting that these peptides are triple chain structures. Quantitative investigation of the Cterminal end of these peptides revealed both lysine and arginine as Cterminal residues. Additional evidence in support of the three-chain postulate was obtained from titration studies. Assuming a molecular weight of 360,000 gm/mole for the original collagen molecule, a n average chain length of 18 residues was deduced from titration of the carboxyl groups liberated on enzymatic cleavage. On the other hand, a n average length of 60 residues was estimated from quantitative amino acid analyses of the separated peptides. It was also observed that addition of leucine aminopeptidase to some of these isolated peptides resulted in the concurrent liberation of several amino acids a t about the same rate. This result is also consistent with the presence of multichain peptide fragments. These studies furnish strong evidence for the existence of relatively stable interchain linkages in collagen. Since the currently accepted view suggests that the collagen molecule is composed of three polypeptide chains, and a t least in certain collagens it seems established that these chains can be largely separated from one another by gelatinization, elucidation of the chemical nature and distribution of interchain cross-links will be of fundamental importance to our understanding of the structure. We shall defer a detailed discussion of this question to Sections IV and V, but it is important to note a t this point that the degree of cross-linking which has been observed in soluble collagens seems to vary with the source of the material studied. For example, results to be presented in Section I V suggest that the individual polypeptide chains of soluble calfskin collagen are more highly cross-linked than those derived from the collagen of the swim bladder of the carp. Furthermore, recent studies relating the degree of solubilization of collagen to the age of the connective tissue suggest that the degree of cross-linking increases progressively with age. Additional work is needed to elucidate the relative structural importance of cross-
TABLE VIa Peptides Derived from Tryptie Digation of the Soluble Collagen of Calfskin >
u -
G
*
z & g z
- & , . g
- - _ _ - ~ ~ -
N-Terminal C-Terminal $!!
Molecular weight by end-group determination Molecular weight by amino acid analysis Mole %, glycine Mole %, Pro. HYPro
I
:
z
& F s "
-__
Gly Gly Gly Gly Gly Gly GlY Gly Gly Gly Arg Arg Lys Arg 2 Lys 2 Lys 1 Lys Lys Arg 2 Lys 1 Arg 1 Arg 2 Arg 1 Arg 1700 1100 1200 2400 2300 l#)o 2300 - 3350 870
Gly Arg
1321 6528 5528 3468 2499
10035 4354 5234 9013
38
-
33 26
33 25
32 24
39 11
4915
3330
36 22
34 17
Gly Gly Gly Gly Gly Gly Gly Arg Arg 1 Lys 2 Lys Lys Arg 2 Lys 1 Arg 2 Arg 1 Arg 4650 - 1600 3000 1700 - - 1420
4630 6936 7722 32 261
30 131
33 24
I
34 191
34 241
34 191
33 26
5840 34 14
591: 368
24 11
33
-
4670
34 25
a Taken from the work of Grassman et al. (1960). Column headings give the specific peptide assignment of the authors for various acid (S),neutral (N), and basic (B) peptides.
STRUCTURE OF COLLAGEN AND GELATIN
41
links between the individual polypeptide chains of a single collagen molecule, and those between adjacent molecules in the connective tissue matrix. b. Collagenolytic Digests. The isolation and purification of a n enzyme from the culture medium of Clostridium histolyticum (Gallop et aE., 1957b; Seifter et al., 1959; MacLennan et al., 1953; Mandl et al., 1953; DeBellis et al., 1954; Schuytema and Kallio, 1956) have provided us with a highly specific agent for the degradation of the polypeptide chains of collagen. Although collagenase attacks collagen and gelatin readily, no other protein so far tested has been found to act as substrate. This remarkable proteolytic property has stimulated a number of laboratories to investigate the specificity requirements using a variety of low molecular weight synthetic substrates. It appears from these studies (Seifter et al., 1959; Michaels et al., 19.58; Nagai and Noda, 1959; Heyns and Legler, 1959; Kazakova et al., 1958; Grassmann et al., 1959; Nagai et al., 1960; Poroshin et al., 1960) that enzymatic activity requires the general sequence -Pro.X.Gly.Pro- with cleavage occurring between the X arid Gly residues. Both proline residues can be replaced by hydroxyproline, but Gallop and Seifter (1961) report activity is substantially depressed by such substitution for the second proline group. Collagenase hydrolyzates of calf collagen and ichthyocol (derived from carp swim bladder) are found to contain peptides in which the C-terminal residue (X) is virtually any amino acid; however, the N terminal residue of these same peptides is always glycine with perhaps one exception. Preliminary studies indicate that this peptide may have an N-terminal alanine (Gallop and Seifter, 1961). A few exceptions to the general rule have also been observed in synthetic substrates. Nagai et al. (1960) have reported that Gly.Pro.Leu.Gly.Gly.Pro is cleaved between leucine and glycine, while Heyns and Legler (1959) and Kagai et al. (1960) have demonstrated carbobenzoxy-Ala.Gly.Pro.-NH2 to be split between Ala and Gly. The rate of splitting these peptides is markedly lower than that observed for the general sequence. Most of the peptides liberated from collagen after digestion with collagenase have an average peptide weight of approximately 500-600, but a number of larger peptides are also released which are too massive to pass through a dialysis membrane (von Hippel et al., 1960). Schrohenloher et al. (1959) have succeeded in isolating and identifying two peptides which occur in relatively large amount, following digestion of steer hide collagens with a crude collagenase extract. The two tripeptides were identified as Gly.Pro.Hypro and Gly.Pro.Ala. Together they accounted for 23 % of the alanine, 14 % of the glycine, 23 % of the hydroxyproline, and 40% of the proline of the original substrate. Essentially the same results were obtained by Gallop and Seifter (1961) using a highly purified collagenase.
42
HARRINGTOR' AND VON HIPPEL
IV. THESTRUCTURE OF COLLAGEN
A . Structural Studies in the Solid State I n this section the structure of collagen in the solid stat,e will be considered a t two levels of magnification. First we will describe in detail the development, of current ideas on the configuration and packing of the polypeptide chains in collagen, and the wide-angle X-ray diffraction and infrared absorption data upon which these ideas depend. Then cert,ain aspects of the larger-scale structure of the collagen fiber, as viewed by elect.ron microscopy and small-angle X-ray diff ractioii, will be discussed more briefly.
FIG.8. Wide-angle X-ray diffraction patterns of collagen from rat tail tendon: ( a ) unstretched; ( b ) stretched 8%. (From Randall, 1954 )
1. The Polypeptide Chain Configuration of Collagen The main features of the collagen wide-angle X-ray diffraction pattrrn have been known for over 30 years. In fact, as pointed out in Section I, this distinctive pattern has generally been considered the best analytical test for the presenw of collagen in a sample of tissue. The principal reflections charactrristic of this material may be seen in the typical wideangle X-ray diffraction photograph which appears in Fig. 8a. (For a detailed description, see Bear, 1952; or Millionova and Andreeva, 1957a.) The 2.86 A meridional arc and the approximately 11 A equatorial reflections are particularly prominent. Painter arcs may also be seen on the meridian at 9.Y, A and 4.0 A, while a diffuse halo appears at about 4 A near the equator. The -11 A equatorial sparing is very sensitive to hydration, shifting from about 10.4 A in completely dry tendon to an upper limit of 15 to 16 A as the moisture content of the fiber is progressively increased (see Rear, 1952; Rougvie arid Bear, 1953). The wide-angle meridional
STRUCTURE O F COLLAGEN A N D GELATIN
43
spacings are not appreciably affected by hydration. &lore recently, Cowan et al. (1953) showed that stretching the collagen fiber by about 10% during the recording of the X-ray photograph results in a considerably sharper pattern containing many more discrete reflections (Fig. 8b). These “stretched” patterns reveal that the meridional arc a t 4.0 A actually consists of two off-meridional spots, and that the diffuse equatorial reflection a t -4 A also may be resolved into two off-axial intensity maxima, leaving only the 2.86 A arc truly on the meridian and the hydrationsensitive 11 A reflection on the equator. Despite the early availability of reasonably good X-ray diffraction photographs (as fiber pictures go) the structure corresponding to this pattern proved remarkably elusive and only in 19.55 were a pair of structures finally proposed, more or less simultaneously, by Rich and Crick (1955) and by Cowan et al. (1955a), which have been generally accepted as being substantially correct. Most of the early proposals for the structure of collagen were based primarily on an attempt to achieve two objectives: (1) to provide a reasonable explanation of the strong 2.86 A meridional reflection; and (2) to somehow fit the large number of imino acid residues into the structure. Furthermore, it was generally felt that proposed structures should account for the apparent inextensibility of collagen fibers.l On the basis of these criteria, structures were proposed by Astbury (1940), Huggins (1943), Ambrose and Elliott (1951b), and others. For a discussion of these proposals we refer the reader to the original papers or to reviews by Bear (1952) and Kendrew (1954), because in 1951-52 two developments totally altered the situation, making all previous structures obsolete. First, in 1951, Pauling and Corey and co-workers wrote their classic papers specifying stereochemical criteria which any proposed polypeptide-containing structure must satisfy. Then, the following year, Cochran et a2. (19.52) presented their calculations of the Fourier transforms for helical structures, making possible direct examination of wideangle X-ray patterns for the presence of helical configuration. These advances made the problem both easier and more difficult; easier because a discrete structure now appeared possible, and more difficult because the number of criteria which an acceptable structure had to satisfy became much more numerous. The first consequence of these developments was the general realization It should be pointed out that Schmitt, et al. (1942) have shown, under certain conditions in the electron microscope, t h a t collagen fibrils can apparently extend manyfold. This led Bear (1952) to suggest t h a t a successful model should allow for such extensibility. However, under all other conditions collagen fibers have proved essentially iriextensible beyond about 10% over rest-length, and in recent years this requirement for the collagen structure seems to have been generally abandoned.
44
HARRINGTON AND VON HIPPEL
that all of the structures proposed up to that time must be in error, due to more or less scrious violations of the Pauling-Corey criteria regarding bond lengths arid angles and coplanarity of amide groupings. Also, examination of the diffraction pattern on the basis of the results of Cochran et al. (1952) quickly showrd that collagen must be wound into a helical configuration (Cohen and Bear, 1953; Cowan et al., 1953).z In the same series of papers in which they outlined the stereochemical ptable polypeptide structures and presented detailed descriptions of the a- and y-helices and the p-structures, Pauling and Corey (1951~)also proposed a structure for collagen. They suggested a threechain structure with each polypeptide chain coiled into a helix, all three helices having a common axis. In this model equivalent amino acid residues occurred a t the same level in each chain, the a-carbon atoms of the three residues a t a given level occupying the corners of an equilateral triangle located perpendicular to the fiber axis. These triangles werc spaced regularly along the axis, a rotation of 40" and a translation of approximately 2.86 A mnverting one three-residue element into the next. The required repeat of 2.86 A was achieved by specifying a cis-trans-cis sequence of peptidc bonds and a mrrcsponding G1y.Pro.X arrangement of amino acids, where X could bc any residue other than proline or hydroxyproline. Interchain peptide hydrogen bonds were made between two of the three residues in each triad, with the bond direction perpendicular to the fiber axis. No hydrogen bonding between triple-chain elements was proposed, permitting these elements to move relative to one another as suggested by the hydration-sensitivity of the 11 A equatorial spacing. This structure represented an improvement over most of the prcvious attcmpts in that it (naturally) satisfied the Pauling-Corey criteria, was helically wound, aiicl gave a true 2.86 A repeat along the fiber axis. However, in the light of subsequent experience it proved inadequate and had to be abandoned. The following difficulties became apparent: (1) Although the model accounted satisfactorily for the 2.86 A axial spaeing and the behavior of the 11 A equatorial reflection, Randall et al. (1953b) pointed out that the structure did not explain certain other features of the X-ray pattern, nor was it quantitatively compatible with infrared dichroism measurements. (2) Thr model rcquired that two-thirds of the total peptide bonds assume the cis-configuration. Yet careful infrared absorption measurements (Badger arid Pullin, 1954) showed that collagen contains very few, if any, cis-amide groupings. Furthermore, Corey and Pauling (1953) themselves pointed out that the trans-configuration is probably more stable than the 2 An excellent general discussion of the characteristics of wide-angle patterns obtained from helical diffractors is given by Stokes (1955).
STRUCTURE OF COLLAGEN rlND GELA4TIN
45
cis, and suggested that since the cis-configuration had oiily been established unequivocally in a single, rather unusual case (in the cyclic dipeptide, diketopiperazine) , the trans-form of the amide grouping was to be preferred and should be used in proposed polypeptide structures whenever possible. (3) The amino acid sequence work of Schroeder et al. (1953, 1954) and Kroner et al. (1953, 1955) showed that the sequence Pro.Hypro is quite common in collagen; yet this sequence could not be accommodated in the Pauling-Corey structure. I n 1952, Randall and co-workers proposed a quite different st,ruct>ure which met most of the above objections. They placed all the peptide bonds in the trans-configuration (achieving the required meridional spacing by tilting the residues to obtain a projected 2.86 A axial separation between a-carbon atoms) and were able to orient the N-H groups so as to satisfy the then-available infrared data. Furthermore, their model accounted satisfactorily for some of the wide-angle X-ray reflectZionswhich the Pauling-Corey model could not explain. However, the model they proposed was basically a two-dimensional sheet structure, and as such was not in accord with the helical configuration suggested by the general shape of the X-ray pattern. Discarding all previous models, Bear and co-workers began a more systematic approach to the problem of collagen structure (Rear, 1952; Cohen and Bear, 1953; Bear, 1955) .3 Instead of proposing a specific stmct,ure, Cohen and Bear began by laying down a set of conditions which any successful model must satisfy. From an analysis of the wide-angle pat,tern they deduced that, the collagen structure must be helical and consist of seven roughly “equivalent, scattering groups” located along a discontinuous helix making approximately two turns per 20 A rise along the fiber axis. Subsequently, Bear (1955) found slightly better agreement with the X-ray data by assuming ten scattering groups located along three turns of such a “genetic” helix.4 Bear also noted that t.he posit,ional aspects of the diffraction pattern could be satisfied by specifying 2-, 3-, or n-chain 3 A similar, systematic survey of various alternative helical structures for collagen was undertaken a t about the same time by Cowan and co-workers (1953, 1955 a , b). It should be noted t h a t the term “equivalent scattering group” does not necessarily imply single amino acid residues. In fact, for collagen, density considerations suggested approximately three residues (with an average residue weight of 93 g/mole) per equivalent scattering group. Bear (1955) adapted the term “genetic” helix from botanical usage, defining the genetic helix as the single helix with the smallest number of turns per period which could be passed through all the equivalent scattering groups. For a more detailed discussion of these points and the general use of “helixnet” theory in the systematic derivation of polypeptide chain structures from X-ray data, the reader is referred to Bear’s (1955) lucid presentation.
46
HAHHISGTON A N D VON HIPPEL
helical strurtures, a t thr same time increasing the axial period 2-, 3-, or n-fold. However, additional ambiguities exist, so that, as Bear stated : “While the application of transform theory has limited the heliral modcls which may br ent,eriained for collagen, these restrictions are far from capable of isolating n unique structure.” Kevertheless, he felt that a systematic approach based on these principles and a knowledge of the stereochemical properties of polypeptide chains, with ultimate testing of detailed structures by optical diffraction methods, should be able to isolate the correct structures. As it turned out, the apparently successful models were initially derived by others, partly by analogy with the structures of certain synthetic polypeptides (see below and Section 11), but Bear (1956) was able to use the systematic elimination approach to confirm independcntly the essential correctness of these structures. As these ideas were developing, the elaboration of specific collagen structures continued. Considerably earlier, Bear (1952) had tentatively put forward, as a collagen prototype, a slightly modified version of the y-helix described by Pauling et al. (1951). This structure, a single helix with a rather shallow pitch, could a t least account for the apparent sevcralfold extensibility which the electron micrographs of Srhmitt et al. (1942) seemed to require. However, a single helix of this type seemed unsatisfactory in that it could not accommodate imino acid residues, did not account for the perpendicular dichroism of the N-H and C=O stretching frequencies, and did not incorporate a unique explanation of the 2.86 A meridional reflection. I n 1934 Huggins proposed a quite different single-stranded helical structure. The polypeptide chain in his model was coiled in a left-handed helix, with ten residues per turn and a pitch of 9.5 A. It was built assuming l’nuling-Corey bond anglrs and distances and planar amide groupings, and included rectilinear, intrachain N-H. . . O peptide hydrogen bonds of equal length at two out of three residues. However, this model was also based on a three-residue repeating unit, with only one position able to accommodate either proline or hydroxyproline without distortion, again excluding the common Pro.Hypro sequence from the crystalline portion of the structure. Other difficulties included the requirement that half of the peptide bonds he in the cis-configuration; also the density calculated for the structure seemed slightly low. Crick (1954) proposed a two-stranded helix, somewhat reminiscent of the Watson-Crick double-strand helix which had been so successful for deoxyribonucleic acid. Thcb model consisted of two polyprptide chains wound helically around a common axis and held together by interchain peptide hydrogen bonds. The peptide bonds were all in the cis-configuration, and the rcprating unit consisted of a pair of amino acid residucs, one perpendicular arid the other parallel to the fiber axis. As a result, plaiics
STRUCTURE OF COLLA4GES AND GEL.4TIK
47
perpendicular to the fiber axis and passing through the a-carbon atoms of pairs of residues a t each level were equally spaced a t a separation of 2.95 A, close to the 2.86 A spacing observed. However, as Crick himself pointed out, this model incorporated some unusually close van der Waals’ contacts. Furthermore, it suffered from incompatibility with the measured infrared dichroism (Sutherland, et al., 1954), was built entirely from the less favored cis-amide groups, and showed some minor discrepancies when an optical transform, constructed by Wyckoff and Chow (see Bear, 1955) was compared with the experimental X-ray pattern.
FIG.9. Three-chain collagen structure proposed by Ramachandran and Kartha (1954). Dotted lines represent hydrogen bonds. Only one pyrrolidine ring is shown. The center line indicates the direction of the crystal axis. (From Ramachandran and Kartha, 1954.)
Finally, in 1954, Ramachandran and Kartha proposed a structure which, while incorrect in certain particulars, was along the right lines and with suitable modification did lead to the present generally accepted models. I n this structure the residues were arranged in three equivalent polypeptide chains, each wound in a left-handed helix with three residues per turn and a pitch of 9.5 A. While also a three-chain model, this structure differed from that of Pauling and Corey in that each chain coiled around its own axis, rather than around a common axis. The three chains were held together by hydrogen bonds as follows: in each turn of each helix two of the three peptide nitrogens were hydrogen bonded to one of the carbonyl oxygens of each of the other two chains (see Fig. 9). This model had a
48
HARltINGTON A N D V O S HIPPEL
number of positive features: all amide groups were built to the PaulingCorcy dimcrisions aiid in the trans-configuration, hydrogen bond lengths were (.lose to the accepted values aiid the N-H arid C=O groups were oricrited so as to agree, both qualitatively and quantitatively, with the infrared dirhroism measurements of Sutherland et al. (1954) ; (see Ramaehandran, 19ri.5). However, it still left unresolved some rather formidable difficulties. First, it was clear that an arrangement of several polypeptide chains with axes parallel to the fiber axis and to one another required that the apparently meridional 2.86 A arc actually arise from the superposition of two, somewhat off-axial, intensity maxima. Using the older, rather fuzzy X-ray patterns, this possibility could still be entertained, but with the advent of the stretched-fiber wide-angle patterns of Cowan et al. (1953) it became increasingly clear that the 2.86 A reflection was truly meridional, and must be accounted for as such by a successful model. Also, the structure as given incorporated hydrogen bonds with much larger angles between the ?;-H and the N . . .O directions than generally considered reasonable. And furthermore, as in several previous models, stereochemistry required a G1y.Pro.X rcpeating sequence (with X any residue other than proline or hydroxyproline) ; thus again excluding the common sequence Gly.Pro.Hvpro from crystalline regions. Ramachandran and Kartha soon recognized some of these difficulties, and accordingly published a somewhat modified model in 1955. They found that the first two objections caould be rrctified by simply twisting the thrce straight he1icc.s of thcir previous model into a three-membered, right-handed super-coil winding around the fiber axis. This modification placed the 2.86 ,4 reflection hack on the meridian, and also brought the peptide hydrogen bonds closer to linearity. A t the same time it did not, introduce much distortion into the original minor helices siiicc the major helix coiled only very g r a d ~ a l l yIn . ~ this structure the super-helix repeated itself after thirty residues (per chain), over a fiber axis repeat distance of 85.8 A. The chains were symmetrically disposed around the fiber axis; a rotation of -108” :md a translation of 2.86 A bringing one chain into coincidence with the next. However, this revised model still failed to accommodate the Pro.Hypro sequence, and, as Rich and Crick (1955) soon pointed out, also contained some uncomfortably short van der Wads’ contacts. A few months after Ramachandran and Kartha’s model appeared, Rich The terms “major” and “minor helix” are used here as defined by Crick (1953) in his original presentation of the coiled-coil (or super-helix) concept. The minor helix defines the turns of the residues of an individual chain around its own axis. The major helix define6 the turns of the minor helix around an axis outside of itself: e.g., the common axis running up the center of the group of three chains in the collagen case.
STRUCTURE O F COLLAGEN AND GELATIN
49
and Crick (1955) presented a still further modified version of this basic: structure. Starting from their previous work on polyglycine I1 (Crick and Rich, 1955; discussed in detail in Section 11) they showed that a bundle of three chains, selected from the polyglycine I1 lattice in either of two ways and twisted into a right-handed coiled coil similar to that of Raniachandran and Kartha, resulted in the generation of a pair of structures which were stereochemically entirely satisfactory and which would accommodate the Gly.Pro.Hypro sequence, These structures have been generally accepted as rather close approximations to the structure of collagen in the diffracting regions of the fiber (but see Ramachandran et al. below) and will be discussed in some detaiL6 The development of the two structures may be visualized in two steps, as follows. First, in both structures, the individual polypeptide chains are coiled into a helix with a threefold, left-handed screw axis; movement along a single helix from one residue to the next requires a rotation of -120" and a translation of 3.12 A. Thus each complete turn of an individual (or minor) helix requires three residues and a 9.4 A rise along the fiber axis. As pointed out above, this is basically the backbone proposed by Crick and Rich for polyglycine I1 and for poly-L-proline I1 by Cowan and McGavin, 1955 (see Section I1 and below). Two such chains are shown lying side by side in Figs. 1Oa and lob. The over-all collagen structure may be developed by taking three such chains and setting them parallel so that, viewed from above, the axes form the vertices of an equilateral triangle with sides about 5 A in length. The three chains are also arranged so that equivalent elements (e.g., the a-carbon of residue 1) are at the same level. At this stage, the three chains are related by a threefold screw axis running up the center of the group. Considering now the two chains with axes located in the plane of the page (Figs. IOU and b), it is clear that the third chain may be added by placing it either behind (Fig. 10c) or in front of the other two (Fig. 1Od). In either case it may easily be seen that only one out of every three residues along a single chain (residue 1 in the numbering system of Fig. 10) lies near the middle of the three-chain structure, while the other two lie on the outside. Therefore, in each of the two arrangements of chains described above, only one relative orientation can be found in which every third backbone N-H can make a stereochemically proper hydrogen bond with every third C=O of a neighboring chain. Thus in both structures each three-residue repeating element is hydrogen bonded to each of the other chains via one hydrogen 6 The description of collagen structures I and I1 which follow are adopted in part from a lucid discussion of these models by Rich and Crick (1958). A complete account of these structures, including the details of the derivation of the final models and complete sets of atomic coordinates has recently been published (Rich and Crick, 1961).
50
H.4RTZINGTOS rlND VON HIPPEL
bond, contributing ail K-H to one bond aiid a C=O to the other. Vicwed end-on, the interrhain hydrogen bonds form the sides of the equilatcral triangle mentioned above. Figures 1Oc and d illustrate these points and also show the essential difference bctwecn the two collagen structures. Adding the third chain
a
b
C
d
e
FIG. 10. ( a ) Two polypeptide backbones shown side-by-side; each helically wound, with a left-handed threefold screw axis (vertical lines). The dotted lines between the two chains represent hydrogen bonds. ( b ) Simplified version of Fig. IOU; only C,atoms are shown. (c) Same as Fig. 106, but with a third backbone added behind t h e other two. This arrangement is related to collagen I. The residues are numbered as in Table VII. ( d ) Same as Fig. l o b , but with a third backbone added in front of the other two. This arrangement is related to collagen 11. ( e ) Showing the deformation of the axis of the minor helices of Fig. 1Oc and d to give the compound collagen helix. Note t h a t the axes of the three polypeptide chains now follow gradual right-handed helices rather than running straight. The broken line represents the common axis around which the axes of the three chains wind. (From Rich and Crick, 1958.)
behind the other two leads to a structure related to collagen I (Fig. LOc). Adding the third chain in front of the others leads to a structure related to collagen I1 (Fig. IOd). Thus these structures differ basically only in the way the chains are placed relative to one another. The actual collagen structures may be derived from these imaginary constructions by siniply twisting the latter slightly so that the axes of the individual chains coil slowly about one another in a gradual right-handed
STRUCTURE OF COLLAGEN A N D GELATIN
51
(major) helix (see 1;ig. 1Oe) instead of running straight and parallel. This deforms the threefold screw axis which previously related the chains to one another in such a way that a rotation of -108” and a translation of 2.86 A takes one from a residue on one chain to the corresponding residue on the next. After three such operations one arrives back on the polypeptide chain from which one started, but three residues higher up (see Fig. 1Oc and d ) . Thus, as in the Ramachandran and Kartha structure, one complete turn of the super-helix contains thirty residues per chain and requires an 85.8 A translation along the fiber axis. It should be emphasized again that the original helices (here the left-handed polyglycine I1 or poly- proli line I1 backbone) are only slightly distorted in being incorporated into the major helix.? Having assembled the backbone structures of collagens I and 11, specific amino acid side chains may be added. I n particular, the incorporation of the Gly.l’ro.Hypro sequrnce (which contributed to the downfall of so many previous models) must be considered. Table V I I summarizes the possible positions of various types of side chains in terms of the residue numbering system used in Fig. 10. The year 19.55 seems to have been a vintage year for collagen structures. Only 3 weeks after the publication of the Rich-Crick models, a paper appeared by Cowan, McGavin, and North in which they described essentially the same structures, deduced independently and from a different point of view. As mentioned above, some years earlier the King’s College group (like the Massachusetts Institute of Technology group) had launched a systematic attempt to fit various types of helical systems to the observed wide-angle X-ray diffraction pattern of collagen. By 1953, they had reduced the number of feasible alternatives to a few major types (see Cowan et al., 195.ib; Cowan et al., 1 9 5 5 ~ )iiicluding a three-chain, coiled-coil structure much like that proposed by Ramachandran and Kartha (195.5). Then Cowan and McGavin worked out the structure of poly-L-proline I1 (see Section 11) and it soon became apparent, that an acceptable collageii structure could be built by twisting a group of three chains, each originally folded in the poly-L-proline I1 configuration, into a super-coil. I n attempting The similarity of these structures to t h a t proposed by Ramachandran and Kartha (1955) is striking, and therefore it is worth pausing t o point out just where the dif-
ferences lie. Both models (considering the Rich-Crick btructures I and I1 together) are similar in containing minor helices built on a threefold left-handed scIew axis of essentially identical pitch, and both feature identical right-handed major helices. The models differ basically only in the way the three chains are packed together; Ramachandran and Kartha attempted t o form t wo systematic interchain peptide hydrogen bonds per three-chain segment, while Rich and Crick were content t o form only one such bond per three residues in order t o achieve a stereochemically more satisfactory structure.
52
HAKKINGTOS AND VON HIPPEL
to build this stru(Ature, Cowan et al. also discovered that the sequcnce Gly.Pro.Hypro could only be accommodated by limiting themselves to one peptide hydrogen h i d pcr three-residue element, and that, on this basis, two acceptable modcls could be constructed. These two structures are TABLEVII Possible Positions of Side-Chains i n Collagens I and IIa Collagen I
Position Undeformed
Collagen I1
Deformedb
1
Gly only
Other residues may be possible; Pro or Hypro impossible
Gly only
2
Any residue including P r o or Hypro
Any residue including Pro or Hypro
Any residue including Pro or Hypro
3
Gly only
Any residue, including Pro or Hypro, exrept Valine
Any residue, including Pro or Hypro
Can make a hydrogen bond t o the neighboring chain within the group of three chains
Projects rrtdially away from the structure and cannot make a hydrogen bond within the group of three chains
__ ~
Bonding of the OH of Hypro in position 3
Position of the interchain hydrogen bonds
From the NH of residue 1 t o the CO of residue 1 on a neighboring chain
From the NH of residue 1 t o the CO of residue 2 on a neighboring chain
From Rich and Crick (1958). Side-chains could be added much more easily t o structure I by permitting certain small deformations of the backbone structure, so allowed side-chains for a deformed structure I are also listed. a
b
equivalent to structures I and 11 of Rich and Crick, but were called the “antklockwise” and “clockwise” structures, respectively, by Cowan et al. (corresponding to the direction of the NH. . .O peptide hydrogcri bonds, viewed from the carboxyl end of the chains). Shortly thereafter, Ramachandran (1956) reconsidered his original proposals on the hahis of the work of Rich and Crick, and Cowan el al., and
STRUCTURE O F COLLAGEN AND GELATIN
53
also agreed that thta Ricbh-Crick structures were probably, in general, correct.* And when Bear (1956) announced the elimination of the other theoretically possible models via a complrtrly indrpeiident, systematic model-building approach, the general validity of the Rich-Crick structures seemed rather well rstablished. Most of the recent wide-angle and infrared work on collagen has been aimed at determining which of the two proposed structures is actually the more nearly correct.$ This is difficult, since the two models actually differ too slightly to be definitely distinguished on the basis of the available data. In fact, the possibility that stretches of both could exist in the collagen fiber should not be entirely dismissed. However, what evidence there is seems to favor the collagen I1 structure. Ramachandran (1956), Burge et al. (1(358),and Rich and Crick (1958) all prefer structure I1 because it is stereochemically more satisfactory and does not require deformation to admit the Gly.Pro.Hypro sequence. Bear (1956) obtained optical transforms of both structuresand found structure I1 in slightly better agreement with the X-ray pattern. The King’s College group carried out a detailed comparison between the diffraction pattern calculated for collagen I1 and the observed wide-angle diffraction pattern, and found reasonably good agreement (Burge et al., 1958; Bradbury et al., 1958). I’auling (1958) built both structurrs using high precision, dural molecular models and found the identity period in collagen I1 to be rxactly 28.6 A, as required by the diffraction pattern. Collagen I was less satisfactory in this respect. However, both models were compatible with the infrared dichroism data. In 1958, Beer et al. re-examined the infrared dichroism situation and remeasured the dichroic ratio for several of the peptide bands in collagen. On the basis of studies on model compounds, they calculated the directions of the transition moments for the principal bands in the peptide link and, using the atomic coordinates for various proposed collagen models, the inc.lination of these transition moment vectors to the fiber axis. Since Beer (19Fi6) had shown that the dichroism of a partially oriented polymer may be considered equivalent to that of a sample containing fully aligned arid completely random portions, a “disorientation parameter” (f) characteristic. of the degree of order of the sample could be calculated for each band. The “spread” of the values obtained for this parameter, which is characteristic of the &leonly and not of the bands used to determine it,
* Raniachandran called the two models (‘plus” and “minus,” corresponding t o the Rich-Crick structures I and 11, respectively. For simplicity, in t h e remainder of this review w e will use the Rich-Crick nomenclature. 6 Certain more indirect evidence bearing on this question comes from physicochemical and infrared studies on interchain hydrogen bonding involving the OH group of hydroxyproline, since the orientation of these groups is markedly different in collagens I and I1 (see Table VII). These studies will be considered below.
54
HAIZRINGTON . W l ) V O N HIPPEL
rcprcsrnts a quantitative niwsure of the compatability of the obsrrvcd iiifrarcd dic*hroismand the proposed strurturr. Perfect agreement should, of course, lead to identical values off for each band. Brrr and co-workcrs used this inethod t o trst three proposed collagrn models: t,hose of I’auling and Corey (1951c), Iiamachandraii and Kartha (IS.%), aiid Rich and Crick (1955) (collagen 11). The Ramachandran-Kartha structure gavr the best agreement of the three; the Rich-Crick structure was somewhat less satisfactory. However, Beer et al. pointed out that the latter structure should wrtainly not h r rcjrctcd 011 this basis alone. The I’auling-Corry structure was found definitely unarceptable, in accord with the X-ray rrsults. The structurrs proposed by Rich and Crick and by Cowan, McGavin, and North, havr not attained completely universal a( ptancr. Huggins (18.57) stated that, in his opinion, there was little rhance that these models could be even approximately correct, because: “. . . they conform neither to the principle that, hydrogen honds are formed whenever possiblr nor to the principle that like groups tend to be surrounded by closr neighbors in a like mannrr. Also. . . there seems to be no way of explaining the large exteiisioiis of the band pattern occasiorially observed in thr elecatron mirroscopr pictures.” Huggins then presented a siiigle-chain hrliral model somewhat similar to his previous proposal (Huggins, 1954) but, improved in that it now cwitaincd only trans-amide groupings. As in his prr\‘lous ‘ model, thr polypeptidr chain was coiled into a left-handed hrlix with thirty residues (ten equivalent scattering groups) per three turns, arid per 28.6 A risr along the fiber axis. In the 1957 modrl, each amino group of the peptidc linkage was hydrogen bondrd to a carboxyl oxygen locaatrd three residucxs further down thr chain, again prrmitting the formation of two intrachain peptidc hydrogen bonds per thrrr-rrsidue unit. As before, oiw could ol~jerito this model on the grounds that the stereochemistry required thc repeating sequrncc GIy.Pro.X, with X neither prolinc. nor hydroxyprolinc. IIowever, Huggins stated that these residues could he placed in position X wit>hout,murh disruption of the structure. IKIdiscussing this model, l’auling (1958) pointed out, that, he and Corey had also proposed an identical singlc-chain structiirr, but had found on attempting to huild a prrcisr model that the stable configuration actually contained four uiiits per turn rathrr than the 8.33 suggested by the X-ray data. Icor this reason arid hecausr of the difficulty in fitting the Gly.Pro.Hypro se(1uencr, I’auling felt that this model must be abandoned. Paulirig (1 958) and cdleagues also used the precise model-building approarh to produce rcrtain stereochemically feasible variants of the collagrri I aiid 11 structures, in which one amide group in csch of the thrccresidue elements of collagen had lieen rotated by 180” about thc single bonds coiinrcting it to the adjacent carbon atoms. Howevrr, the “reversed”
STRUCTURE O F COLLAGES AX D GELATIN
55
structure corresponding to collagen I1 gave the wrong identity period, while reversed structure I gave the proper identity period but could riot accommodate the Gly.I’ro.Hypro sequence. Thus, from all points of view, the collagen I1 structure seemed to represent the best available model for the configuration of the polypeptide chains in the crystalline portions of the collagen fiber. Very recently, several long papers dealing with the structure of fibrous proteins and polypeptides have emanated from the laboratory of G. hi. Ramacharidran (Ramachandran, 1961; Ramachandran et al., 1961; Lakshmanan et al., 1961 and Sasisekharan, 1961).1° Specifically, some new ideas on collagen structure are included which seem worthy of careful consideration. Though final evaluation of the new data and suggestions contained in these papers must await the passage of time and consideration by workers involved directly in the field of fiber X-ray diffraction analysis, we will attempt to examine some particularly pertinent portions of this very comprehensive work. On the basis of an extensive compilation of bond lengths, angles, and unbonded contact distances derived from various organic Structures which have heen measured accurately, Sahisckharan emphasized that : (a) primary valence bond lengths and angles may deviate appreciably from standard values; (b) hydrogen bond lengths and angles differing markedly from the “normal” values are often found (see also k’uller, 1959, for a similar recent compendium of hydrogen bond dimensions) ; and (c) UHbonded contact distances somewhat shorter than the sum of van der Waals’ radii can occur. Moreover, known structures do not always exhibit complete amide-group coplanarity. Sasisekharan classified these various parameters into groups designated “common,” “occ.asiona1,” arid “rare,” depending upon the frequency with which they occur in known structures. On the strength of this compilation, Ramacharidran et al. have questioned the rigidity with which stereochemical parameters have been applied in formulating and defining acceptable structures for proteins and polypeptides. Though individually the standard values doubtless represent the energetically most favorable situation in each case, it is clear that compromises must often be made in individual param rs to achieve maximum stability of the over-all structure. Therefore structures should not, a priori, be discarded because they do not satisfy the ideal Pauling-Corey criteria completely, especially if the suggested distortions fall within the ranges observed in known structures and if additional, previously excluded, modes of stabilization thus become available. The present, generally accepted model for collageii (collagen 11) has 10 We are grateful t o Professor Ramachandran for sending us manuscripts of these papers prior t o publication.
56
HAHRINGTOS AND VON HIPPEL
heen re-c.xamined iii the light of thcse ideas and some new X-ray studies (wried out, by Lakshmanan ct at. Considering first the collagen I1 strurturc (which they term the “single-bonded 11” structure, referring to the fact that this structure forms only one peptidc hydrogen bond per three-residuc unit) Ramacbhandran and co-workers point out that this structure is stereochemically far from unique. By varying the various dimensional parameters systematically through the common range, a wide variety of triple-chain, single-bonded structures may be generated, with no one structure particularly stereochemically preferable to another. Thus there appears to be no stereochemical rcason why such a single-bonded structurc should assume the particular helical parameters characteristic of collagen 11. In fact, Ramachandran et al. show that a satisfactory single-bonded structure can be made without invoking a coiled coil, without requiring that every third residue be glycine, and with projected axial residue repeats varying withiii rather wide limits. On thr other hand, by permitting a few short van der Waals’ contacts, a triple-chain struct lire with two interchain hydrogen bonds per t hrecresidue chain element can be coiistructed. This structure, which Ramachandran ct at. refer to as the standard double-bonded structure, is actually only slightly different from that previously put forward by Ramachandran and Kartha (195.5). All bond angles and distances, uiiboiided eontact distances, and hydrogen bond dimensions are held within the observed (though riot always the common) ranges, and therefore Ramachandran el aE. propose that this structure he considered satisfactory; the extra strain energy resulting from slight dwiations from the “standard” values of tht. stereochemi d parameters should be more than compensated by the stabilizing influence of the second hydrogen bond. This structure actually fits the X-ray evidence more closely than the single-bonded I1 structure (see Itamachandran et al. 1961; Lakshmanan et al., 1961) and also provides better agreement with the infrared dichroism results (Beer ct al., 1958). Moreover, the double-bonded strwture is highly specific and can accommodate only helical parameters close to those which actually occur in collagen, thus offering a natural stereochemiral explanation for the specific coiled-coil structure ohserved and for the requirement that every third residue be glycine. As beforp, only oiie member of the three-residue repeating chain can be an imino acid without deforming the structure, so again, ideally, thc sequence Gly.Pro.Hypro is excluded. However, only a slight (and stereochemically permissible) adjustment need be made t o accomrnodatc this sequence. Figure 1l a shows a projection down the axis of the proposed double-bonded structure, whilc Fig. 110 shows how this structure may be modified to accommodate the Gly.€’ro.Hypro sequence. It should be noted that if only one of the three chains a t a given level contains this sequence, then only
FIG.11. Axial projection of three polypeptide chains cast into the “doublebonded” structure proposed for collagen by Ramachandran et d.(1961). The dotted lines represent hydrogen bonds. ( a ) The standard structure; ( b ) structure modified to accommodate two imino acid residues per three-residue repeating unit. The undistorted position of the B chain is also shown (lightly) t o indicate the extent t o which the various atoms of the chains have been moved relative t o the undistorted structure. (From Ramachandran et al., 1961.) 57
58
HARR1NC:TON AND VON HIPPEL
that chain need adopt thc modified configuration and five iriterchairi hydrogen bonds can still be formed per iiiiie residues. As a (wiisequeiiw of these developments, we must close this sectioii on a slightly less definitive note than might have been possible a few months ago, stating only that there seems, at least, to be general agreement that collagen is a triplc-chain coiled-coil structure stabilized by one or two interchaiii pept ide hydrogen bonds per three-residue repeat>ingm i t .
2 . The Structure of the Collagen Fibril Turning now to the larger-scale features of the collagen complex, we must consider first what fraction of the polypeptide chains is actually cast into the ordered configuration drscribed above. Since the crystalline portions give rise to the discrete reflections of the widc-angle X-ray pattern, while the amorphous (nonrcpeating) regions are responsible for the difiuse background, one should in principle be able to calculate the fraction in each form by comparing intensities in appropriate portions of the pattern. I n practice this is difficult and one must turn to other methods t o invcstigate this question ; specifically, in this case, to electron microscopy and small-angle X-ray diffraction. Vicwed microscopically, collagen appears to he laid down in \midles madc up of parallel fibrous elements. These fibcrs seem smooth and undifferentiated at thc resolution of the light microscope, but as the early c~lcctron microwope work of Wolpers (1043, 1944) and especially of Srhmitt and co-workers (1942, 1945), Schmitt and Gross (1948) showed, the individual fibrils which make up the collagen fiber are characterized by a distinctive pattern of fine cross-striations. At high resolution, in suitably stained prcparations, these striations are seen to consist of regularly repenting srts of “baiids” and “interbands” having fixrd rclative positions and electroil densities. In native fibrils, the over-all pattern repeats s t an average axial interval of about 640 A (close to 700 A in hydrated samplcs). Figure 12 shows a typical elec%ronmicrograph of stained fihrils at a fairly low magnification; the interband structure of the repcating unit may be setn in Fig. 13, which shows a greatly enlarged (and schematized) view of one ti40 A period of native collagen. It should be noted that this intraperiod band pattern is polarized; i.e., it is not symmetrical about a plane passed through the centcr of the period normal to the fiber axis. This intraprriod finc structure is not always observed, but a regular alternation of slightly enlarged, phosphotungstic acid-staining bands, mid smaller diameter, nonstaining interbands is characteristic of almost all of the native collagens which have been investigated. At ahout the time that collagen was first examined with the electron mirroscope, Rear (1942, 1944) and Kratky and Sekora (1943) succcedcd
STRUCTUHE O F COLLAGEN AND G ELA TI N
59
in resolving the small-angle X-ray pat tern characteristic of this material (Clark et al., 1935; Wyckoff et al., 1935) and showed that all the layer lines which appeared spaced along the meridian of the pattern (see Fig. 14) represent higher orders of a fundamental axial repeat of about 640 A. Using special cameras and finely collimated beams, as many as thirty diffraction orders have heen observed in carefully prcpared specimens (Bear et al., 1951; Kaesbcrg and Shurman, 1953; Tomlin and Worthington, 1956). Thus
FIG.12. Electron micrograph of air-dried, chromium-shadowed collagen fibrils from adult human corium; magnification. X 2G,000. (From Gross and Schmitt, 1948.)
the large-period repeating structure which gives rise to these lincs is obviously highly ordered. No evidence of regularity prependicular to the fiber axis has been ohserved in small-angle X-ray diffraction studies. Since a fundamental axial repeat of -640 A could be observed by means of both electron microscopy and X-ray diffraction, it appeared that this spacing was not ttrtifactual but represented a definite repeating structure of some kind. Bear (19.52) suggested that the band-interband repeat seen in electron micrographs might bc due to a regular alternation of groups of amino acids, the band regions containing high concentrations of long-chain, polar amino acids and the intcrbands containing mostly the smaller, non-
60
J-IARRINGTON A N D VON I-IIPPEL
polar residues. He proposed that the interhands form the portions of the fibril which give rise to the wide-angle X-ray pattern, the polar amiiio acids located in the bands being too bulky and highly charged to pack properly. Thus the bands would he the most amorphous portions of the fihril, and
FIG.13. Schematic view of the periodic fine structure (phosphotungstic acidstained) of the repeating elements of various reconstituted collagens: native pat tern, fibrous long spacing (FLS) collagen and segment long spacing (SLS) collagen. (From Schmitt e t al., 1955.)
therefore also more easily penetrat,ed by st'ains. Investjigations of t,he cffccts of tension and hydration on the wide- and small-angle X-ray diffraction patterns (Bear et al., 19.51; Cowan et al., 1955b; Bear arid Morgan, 1957) and cert,ain of t'he amino acid sequence studies carried out by Grassmanri and co-workers (see Section 111) lend considerable support, t o t>hishypot,hesis.
STRUCTURE OF COLLAGEN . 4 m GELATIS
61
More detailed correlation of intraperiod fine structure, as viewed by small-angle X-ray diff raction and high resolution electron microscopy, has been attempted by several groups (see Cowan et at., 1955b; Randall el al., 1955; Burge and Randall, 1955; Bear and Morgan, 1957) without, however, casting significantly more light on t h r molecular factors involved. As it turned out, electron microscope studies of “reconstituted” collagen fibrils have proved much more informative. Much earlier, Xageotte (1927) and others had found that collagen could
FIG. 14. Small-angle X-ray diffraction pattern of kangaroo tail tendon. Layer line indices are indicated. Note the “fanning” seen in b and c. This is indicative of a certain amount of chain distortion which accompanies drying; ( a ) moist specimen; ( b ) after soaking in water for 2 months, then drying under tension; (c) a f k r brief exposure t o water, then drying under tension. (From Bear et al., 1951.)
be dissolved in dilute acid solution, and that, this soluble collagen could then be reprecipit,at’edin fibrous form by appropriate manipulation of pH, salt concentration, et,c. (see below). Furthermore, X-ray (Wyckoff and Corey, 1936) and e l e c h n microscopic (Schmit,t el al., 1942) examination revealed that many of the regularit,ics characteristic of native collagen had reappeared in the reprecipita,ted or reconstituted material. These findings have been greatly extended by Randall and co-workers (Randall et al., 1952; Jackson and Randall, 1953; Randall et al., 1953a, 1955) and especially, in a classic series of papers, by Schmitt., Gross, and Highberger (Highberger et al., 1950,19.51; Gross et al., 1952; Schmit,t et al., 1953; Gross et al., 1954).
Schmitt arid his rollaborators found that by judicious manipulation of the solvent environment they could reprecipitate soluble collagen in a t least five different fibrous modifications. Briefly, they found that>by adding increasing quantities of salt to a dilute acetic acid solution of soluble collagen, t,hey could produce reconstituted collagens which showed : (I) the iiormal -640 A polarized banding at 1 % NaCl, (2) an abnormal, -210 A periodicity at 2 76 NaCl, and (3) no periodicity at 5 76 NaCl (see Fig. 15).
FIG.15. Schematic two-dimensional view of various modes of aggregation of rollagen molecules in vilro, illustrating the generittion of the various periodirities. The stagger responsible for the -210 A and the -6-20 A periods is not necessarily all in adjoining molecules, as shown here. (From Schmitt, 1956.)
Furthermore, if instead of salt they added small amounts of acid glycoprotein (or a variety of other agents; see Gross et al., 1952; Randall et al., 1952) the collagen precipitated in the so-called “fibrous long spacaing” (FLS) form, with a nonpolarized repeating period of about 2600 A. The addition of certain other agents (in particular, adenosine triphosphate) under slightly different conditions induced the precipitation of collagen as shorter, polarized segments having a total length of about 2600 A; the socalled “segment long spacing” (SLS) form (see Figs. 1.5 and 13). Furthermore, these forms were shown to he completely interconvcrtiblc. Schmitt and co-workers interpreted these results in terms of a hypotheti-
STRUCTURE OF COLLAGEN AND GELATIN
63
cal, polarized c*ollagen molecule which they called “tropocollagen,” and which they deduced should be about 2600 A in length and 15-20 A in diameter. Such long filamentous collagen molecules could then produce the observed patterns by aligning themselves in parallel :in a quart er-staggered arrangement to form the native collagen period, antiparallel with the ends in register to form the FLS pattern, and parallel with the ends in register to produce the SLS segments.” They assumed that the specificity responsible for these discrete interactions must be “built-in” to the molecules via specific and polarized arrangements of amino acids along the polypeptide chains. An independent demonstration of the general correctness of the “tropocollagen concept” came from the work of Boedtker and Doty (1956; see below) who showed by physicochemical means that soluble collagen in acid solution exists as long, relatively rigid, rod-shaped molecules having a length of 2900 A, a diameter of 15 A, and a molecular weight of about 350,000. Shortly thereafter, as a consequence of improvements in electron microscope technique, Hall (see Hall, 1956; Hall and Doty, 1958) was able to obtain electron micrographs of individual collagen molecules. These confirmed directly the dimensions measured by Boedtker and Doty, and deduced (for the hypothetical tropocollagen monomer) b y Schmitt and coworkers. The potent combination of chemical and enzymatic methods and electron microscopy has been used in further studies of the structure and interactions of the collagen molecule. In particular, this approach has been useful in mapping the distribution of different classes of amino acid residues along the molecule. As mentioned above, Bear (1952) originally suggested that the phosphotungstic acid-staining bands observed in the electron microscope represent amorphous regions containing primarily the bulky polar residues of collagen; Kuhn et al. (1957) showed that these regions probably take up phosphotungstic acid by relatively specific chemical combination with basic amino acid residues. Hodge and Schmitt (1960) have carried this analysis further using the SLS-type aggregates, in which all the like features of a series of collagen molecules are arranged in accurate transverse register. Taking advantage of this characteristic, they have been able to use SLS band patterns as “molecular fingerprints” to localize various specifically bound, elect ron-dense compounds, and to deduce the packing mechanisms which apply in other types of ordered collagen aggregates. They examined and compared SLS aggregates stained with: (a) phosphotungstic acid, which reacts with the positively-charged lysine and arginine residues and (b) uranyl ions, which bind to aspartic and glutamic acid 11 For more detailed reviews of these studies see Schmitt et al. (1955), Gross (1956), and Schmitt (1959).
64
HARIZIKGTON AICD VON HIPPEL
residues. These two stains yielded SLS band patterns which differed substantially in the rclative intensities of comparable bands, but resembled one another closely in terms of the relative positions of the bands along the collagen molecule. Thus Hodge and Schmitt concluded, in agreement with Bear, that both the acidic and basic polar residues are located in narrow clusters separated by the nonpolar residue-containing interbands. The enzymatic results of Grassmann and colleagues also tend to bear out this interpretation (see Section 111). Recently, Xishigai et al. (1960) treated SLS aggregates with collagenase directly on the electron microscope grid. They found that collagenase selectively digested away the interband regions, leaving the bands relatively intact. Sirice it has becn established (sce Section 111) that collageriase specifically catalyzes the hydrolysis of the peptide bond between residues X and Gly in scquenccs such as -Z.Pro.X.Gly.Pro.Y-, these results again demonstrated that scqucnces of this sort arc confined primarily to the interband regions, and further idcntifies these regions as the crystalline portions of the collagen fibril.
R. Structural Studies in Solution 1. The Isolation of Soluble Collagen
Zachariades (1000a) was the first to show that collagen could be solubilizcd using fairly mild techniques when he found that apprcciahle fractions of the tail tendons of rats could be dissolvcd in dilute solutions of formic, acetic, oxalic, hydrochloric, hydrobromic, sulfuric, and othrr acids. However, thc significance of this finding did not become apparent until 1927, when Kageotte showed t,hat acid extracts of soluble collagen could be reconstituted into fibers which microscopically resembled native collagen. This similarity has been amply borne out by more recent X-ray diffraction and electron microscope studies (see above). Since 1927, many workers have demonstrated that collagen from a variety of tissues can be brought into solution using dilute acids (e.g., see Leplat, 1933; FaurB-Fremiet and Garrault, 1937; Orekhovich et al., 1948; Gallop, 1955a). Most of the recent physicochemical work on soluble collagen has been done on collagen solubilized by one of several variants of the citrate extraction procedure of Orekhovich et al. (1948). Basically, this procedure involves extraction of minced connective tissue with a dilute citrate buffer a t p H 3.5 to 4.0, followed by removal of the insoluble residue, and dialysis of the extract against tap water or dilute salt solutions. The dissolved collagen is reprecipitated as needlelike crystals, which may be harvested and purified by several repetitions of this cycle. Ovcr the last few years it has been shown by several groups that collagen can also he dissolved in a variety of neutral salt solutions (e.g., see Gross
STRUCTURE OF COLLAGEN AND GELATIN
65
et al., 1955a; ,Jackson and Fessler, 19.55; Gallop et al., 1957a) and in mild alkali (Harkness et al., 1954) ; we will use the generic term “soluble collagen” to refer t o the products of all these mild extraction procedures. A considerable literature has developed which deals with the differences between collagens (and gelatins) extracted from the connective tissue in various ways; this will be reviewed in Section V. For present purposes we need only point out that, to a first approximation, the collagen molecules extracted by these various procedures are essentially identical in terms of size, shape, chain configuration, arid most other chemical and physicochemical properties (e.g., see Gross, 1956; Orekhovich et al., 1937; Mazourov and Orekhovich, 1939, 1960; Jackson and Bentley, 1960).12 2. Physicochemical Studies of Soluble Collagen a. Size and Shape of the Collagen Molecule. Since the existence of discrete units of soluble collagen, capable of reconstituting larger scale native structures, had been apparent since the work of Nageotte, it is somewhat surprising that physicochemical studies of soluble collagen were not undertaken for so many years. Indeed, the first work along these lines was carried out only about 12 years ago by Bresler, et al. (1950), on collagen extracted from rat skin by the citrate method of Orekhovich. Bresler and co-workers reported a molecular weight of about 70,000, and a particle length of about 380 A, on the basis of sedimentation and diffusion measurements. However, the collagen used in these studies was dissolved a t 40°C, which subsequent studies have shown brings about the total thermal denaturation of collagen and its complete conversion to parent gelatin.13This work proved to be the first of a series of physicochemical investigations of various soluble collagens by several groups, including: M’Ewen and Pratt (1953), Mathews et al. (1954), Noda (1955), Gallop (1955a), Orekhovich and Shpikiter (19554, and Peng and Tsao (1956). The results obtained by these and subsequent investigators are summarized in Table VIII. (Data obtained a t relatively high temperatures, which therefore probahly relate to thermally denatured collagens, have been omitted.) These groups obtained widely divergent values of molecular weight for soluble collagen (see Table VIII) but were generally agreed that the native molecule must he considerably larger and certainly much more asymmetric than Bresler et al. had suggested. Also, it soon became obvious (see cspecially Mathews et al., 1954; Gallop, 1955a and Boedtker and Doty, 1956) that mild heating, or treatment with urea, KSCN, etc., resulted in the conversion of these large, asymmetric particles However, it is clear t h a t some of the more subtle interaction properties do vary between soluble collagens extracted in various ways, and even between fractions of an extract of a single type (e.g., see Fessler, 1960). ‘SThe term “parent gelatin,” coined by Statchard et al. (1944) t o designate the ideal, undegraded, precursor gelatin molecule is defined in Section V.
HARIZINGTON ANI) V O N HIPPEL
TABLEV I I I Physicochemical Properties of Soluble Collagen Parameter
Values
Collagen and solvent
Intrinsic viscosity, hl (100 ml/gm)
15 15 13.2 10.5 11.5 12-16 13.5 13.7 13.2
Rabbitskin (citrate, p H 3) R a t tail tendon (acid) Ichthyocol (citrate, p H 3.7) Ratskin (citrate, p H 3.7) Ichthyocol (citrate, p H 3.7) Ichthyocol (neutral salts) Calfskin (citrate, p H 3.7) Ratskin (citrate, p H 3.7) Perch swim bladder (citrate, p H 3.7) Cod swim bladder (citrate, p H 3.7) Ichthyocol (neutral CaCl2) Calfskin (acetic acid) Codskin (citrate buffer, p H 3.5) Ichthyocol (citrate, p H 3.7)
13.2
Partial specific volume, 0 (ml/gm) Sedimentation coefficient s ~ O . l (SvedU berg units)
Translational diffusion coefficient, Oh., (cm*/sec) ( x 101) Refractive index increment, d n / d c (ml/gm) Molecular weight, M (gms/mole) : Sedimentationdiffusion Sedimentationviscosity
Osmotic pressure
15-17 15 12.8 0.705 3.5 2.85 3.15 2.90 3.28 3.0 3.17 0.33-0.5 0.35-0.4
0.189-0.197 0.192 0.187 710,000 700,000 510,000 250,000 352,000 280,000 310,000 400,000
Rat tail tendon (acetic acid) Ichthyocol (citrate, p H 3.7) Ratskin (citrate, p H 3.7) Ichthyocol (citrate, p H 3.7) Calfskin (citrate, pH 3.7) Calfskin (acetic acid) Codskin (citrate, p H 3.5) Rattail tendon (acetic acid) Ratskin (citrnte, pH 3.7)
Ratskin (citrate, p H 3) and r a t tail tendon (acid) Ichthyocol (citrate, p H 3.7) Ichthyocol (citrate, p H 3.7) Rattail tendon (acetic acid) Ratskin (citrate, p H 3.7) R a t tail tendon (acetic acid) Ichthyocol (citrate, p H 3.7) Calfskin (citrate, p H 3.7) Codskin (citrate, p H 3.5) Ichthyocol (citrate, p H 3.7) Ratskin (--)
Refer:nce
It C
a e
f h
3 1 1 1 m
n P
a C
a e
f 3
n P C
e
a
a f C
e C
f 3
P
f 9
-
67
STRUCTURE OF COLLAGEN AND GELATIhT
TABLEVIII-Continued Parameter
Values
Light scattering
3.G-10 X 10' 10-25 X 106 1.67 X lo6 345,000 360,000 0.4-0.5 X lo6 1.4 x 108 350,000
Flow birefringenct and Viscosity
Collagen and solvent
Reference
a a d
Itatskin (citrate, p H 3) R a t tail tendon (acid) Ichtbyocol (citrate, p H 3.7) Ichthyocol (citrate, p H 3.7) Calfskin (citrate, p H 3.7) Ichthyocol (citrate, p H 3.7) Ichthyocol (neutral CaC12) Ichthyocol (citrate, p H 3.7)
f j
rrL
m
f
__
L
D ~
Molecular length and diameter, L and I) (angstronis) : Hydrodynamic
Light,-scattering
Electron microscopy
3700-5200 18-22 6000 2900 12-13. 3500 3000 12.1 2810 87-~60x 103 12 88 X lo3 16 13.4 x 103 13 3100 3100 2820 15 3000 15
R a t tail tendon (acetic acid) Ratskin (citrate, p H 3.7) Ichthyocol (citrate, p H 3.7) Calfskin (citrate, p H 3.7) Calfskin (acetic acid) Codskin (citrate, p H 3.5) Ratskin (citrate, p H 3) R a t tail tendon (acid) Ichthyocol (citrate, p H 3.7) Ichthyocol (citrate, p H 3.7) Calfskin (citrate, pH 3.7) Ichthyocol Calfskin
C
e
f j n P a a d
f j k n
~~
M'Ewen and P r a t t (1953). Mathews et al. (1954) c Noda (1955). d Gallop (1955a). e Orekhovich and Shpikiter (1955a). f Boedtker and Doty (1956). g Peng, and Tsao (1956). (1
Gallop et al. (1957a). Doty and Nishihara (1958). k Hall and Doty (1958). 1 Burge and Hynes (1959a). m von Hippel et al. (1960). Rice (1960). p Young and Lorimer (19GO). a 2
to considerably smaller, more flexihlc, subunits (gelatin, see below and Section V). Most of the quantitative discrepancies and contradictions arising from these studies were resolved by the work of Boedtker and Doty (1956) who carried out a very careful study of soluble ichthyocol collagen in pH 3.7
68
HARRINGTON AND VOX HIPPEL
citrate buffcr (isolated and purifird from carp swim bladdcrs using a proccdure developcd by Gallop, 195.5~~). Bordtker and Doty obtained by several combinations of methods (including light scattering, osmotic presmre, sedimentation, viscometry, and flow birrfringence) a molecular weight (weight average) of 345,000 for the ichthyocol monomer in acid solution. The molecule was found to be rodlike in shape, with a length of about 3000 A and a diameter of 13.6 A. These dimensions have since been confirmed by direct electron microscopic observation of single collagen molecules (Hall and Doty, 1958; Rice, 1960). Also, as pointed out above, these findings were particularly important in that they were in good agreement with the dimensions deduced by Schmitt arid co-workers for the hypothetical tropocollagen monomer, and thus served to establish a bridge between the studies of collagen in solution and in the solid state. A careful examination of Table VIII reveals that thc chief discrcpancy bctween thr results of Boedtker and Dotly and those of the earlier workcrs lies in the light mattering values; the intrinsic viscosities and scdirnentation coefficients dcterminrd by various groups differ remarkably little. This led Boedtker and Doty to investigate thc light-scattering problem very carefully. They found that the difficulty seemed to be due to the presence of small amounts of a large contaminating material-presumably large, loosely bonded aggrcgatcs of collagen molecules-which could only be removed by exhaustive, high speed crntrifugation at vcry low protein concentrations. Such aggrcgates would hc cxpected to have a great influence on light-scattrring dctermiriations of particle weight and size, while a fYecting measurements of [q]arid S ~ Ohardly , ~ a t all. This seems to account for thc high values of the earlier light-scattering results. Subsequent work (Doty and Nishihara, 1958; Burge and Hynes, 195%; Young arid Ilorimer, 1960; Rice, 1960) suggests that soluble collagen monomers derived from a variety of other tissues are generally similar to ichthyocol in over-a11 size arid shape, though frce monomers may br found only in acid solution. I n this connection, von Hippcl et al. (1960) have shown that in neutral 0.5 Ill CaClz, ichthyocol owurs predominantly as small aggregates (approximately tetramers) . Recent iriterpretat ions of wide-angle X-ray diffraction patterns have led t o a three-chain structure for collagen, a t least in the ordered interband regions. This strongly suggested that a three-stranded structure should exist in the collagen monomer as well. Several lines of evidence support this view: (1) Thc average mas-to-length ratio ( M / L ) obtained for ichthyocol by light scattering is 100 avograms per angstrom (Boedtker and Doty, 1956). This ratio is in reasonable agrermcnt with the value of 98 avograms per angstrom rrquircd by thc X-ray data (Bear, 1952).
STRUCTURE O F COLLAGEN AND GELATIN
69
( 2 ) The equatmial reflections of the wide-angle pattern suggest a centerto-cent,er separation of 10 to 15 A (depending on the degree of hydration) for adjacent collagen molecules. This compares favorably with the value of 12 to 15 A derived from studies in solution (Table VIII). (3) Complete deiiat,uration of ichthyocol, either by heating or by treatment with neutral salts such as KSCN (see below) brings about a n approximat’ely threefold decrease in weight-average molecular weight, suggesting that the three strands postulated for the native collagen molecule separate under these conditions (Boedtker and Doty, 1956). (4) Correlation of the kinetics of proteolysis of ichthyocol by the enzyme collagenase with changes in various physical parameters, indicate that the native molecule must be multistranded (von Hippel et al., 1960). However, other data suggest that picturing the collagen monomer as a structure composed of three equivalent polypept,ide chains twisted together as required by the X-ray results, may be an oversimplification. For example : (1) The structure must somehow accommodate the various “unusual” covalcnt bonds (e.g., hydroxylamine-sensitive “ester-type” bonds, y-carboxylglutamyl and r-aminolysyl peptide linkages, see Section V) which have been report,ed in collagen and gelatin and which may possibly be involved in interchain cross-linking. (2) The actual molecular weight of the parent gelatins produced from collagens by mild heating is generally not exactly one-third that of the collagen monomer. For ichthyocol, Boedtker and Doty (1956) obtained a weight-average molecular weight of 138,000, resulting in a molecular weight ratio of collagen to gelatin of approximately 2.5. Earlier, Gallop (1955b) had obtained a molecular weight of about 70,000 for parent gelatin, yielding a collagen-gelatin ratio of about 5. Boedtker and Doty postulated, on the basis of their result’s, that the collagen monomer contains three chains of unequal weight. They suggested several possible arrangements of such chains, including a staggered arrangement with a single dangling chain protruding beyond the rigid three-stranded portion of the molecule at either end. This suggestion has been adopted by Hodge and co-workers in some of their recent studies of rcconstit’uted collagen. An important observation by Orekhovich and Shpikiter (1955b) seems to have led to a partial explanation of the discrepancy between the molecular weights obtained for parent gelatin by Gallop and by Boedtker and Doty. Orekhovich and Shpikit’er found that, parent gelatin derived from rat skin contained two components of differing molecular weight. These components, termed a and p, were subsequently observed in calfskin and ichthyocol gelatin solutions as well (Chun and Doty, 1958; Nishihara and
70
IIAHltISGTON AND V O S HIPPEL
Doty, 1958; Orekhovivh arid (Lo-workerb, 1960; Orekhovich and Shpikiter, lY.58b). Iloty and co-workers also found that the heavier, p-component , could be split by alkaline treatment or prolonged heating into units having the sedimentation characteristics of the a-component. This suggested that the @-componentis held together by a very labile bond(s), and that the molecular weights of -70,000 were probably obtained on samples of parent gelatin in which this bond had already been ruptured. This finding may also account for the progressive decrease in the intrinsic viscosity of gelatin which accompanies long standing at moderate temperatures (Boedtker and Doty, 1956). The bond responsible for these changes is probably of the ester type; it is definitely not a (a-amino) peptide bond, since cleavage results in no release of ninhydrin-positive material (von Hippel, unpublished material). A more detailed discussion of the components of parent gelatin will be deferred to the following section. The point to be made here is simply this: that while the generally accepted, three-strand interpretation of the collagen molecule is probably correct, the details of how the molecule is assembled from its subunits remain obscure. Additional information on the structure of the collagen molecule has been derived from studies on sonicated collagens. Nishihara and Doty (1958) found, from scdimcntatiori and viscosity measurements, that sonic irradiation of soluble calfskin collagen (at a frequency of 9 kilocycles per second) resulted in the progressive fragmentation of the molecules into shorter segments, which, however, seemed to retain the compound-helical structure and rigidity characteristic of the native molecule. Time-dependence studies of the rate of molecular weight decrease suggested that clcavage occurred by nonrandom scission at three particular, rather evenly spaced regions of the molecule. Nshihara and Doty pointcd out that these susceptible regions seem to be separated by approximately 700 A, and thus may bear some relation to the regions responsible for the -G40 A spacing observed in phosphotungstie acid-stained samples of nativc. collagen. This prefereiitial cleavage of the collagen monomer into halves and quarters has been confirmed by Hodge and Schmitt (1958) using electron microscopy. Hodge and Schmitt (19.58) carried these ideas further. They demonstrated that sonication did not impede the lateral packing of the degraded molecules into an SLS-type pattern. On the other hand, the ability to form end-to-end aggregates of the native type disappeared rapidly, even before the molecular lengths had been appreciably altered. This suggested that the ends of the collagen molecule must be particularly susceptible to sonic damage, and that such damage destroys the normal md-to-end interaction specificity of the molecules. On the basis of these results arid the
STRUCTURE OF COLLAGEN AND GELATIN
71
collagen model with dangling terminal peptidc chains proposed by Boedtker and Doty (1956), Hodge and Schmitt postulated that formation of the native-type pattern might involve a specific type of coiling of these terminal chains about one another to form a highly ordered structure, and that these terminal chains must therefore be particularly susceptible to destruction by sonic irradiation. In order to test this hypothesis, Hodge et al. (1960) attempted to isolate and characterize these terminal chains by treating intact collagen molecules with trypsin. They found, as had been previously reported by Gallop et al. (1957a), that the native collagen molecule is essentially impervious to tryptic attack. However, at t,he temperature of their experiments (room temperature), some small amount of proteolysis did occur, which seemed to have no effect on the monomer lengt,h, but, like sonication, essentially abolished the ability of treated samples to reconstitute native-type fibrils. This suggested that t,rypsin also attacks, primarily, the end regions of the molecule, perhaps cleaving the postulated terminal peptide chains. An acidic, tyrosine-containing peptide, which may perhaps be part of such a structure, has been isolated by curtain electrophoresis from such tryptic digests. Certain additional, more indirect information on the structure and properties of the collagen molecule may be derived from an examination of the kinetics of the formation of fibrils from collagen solutions. In the studies t o be discussed, fibril formation was initiated either by: (1) diluting an acidic or neutral salt solution of soluble collagen with low ionic strength buffer adjusted to pH -7, or (2) increasing the temperature of a neutral salt solution of collagen to 37°C. The latter procedure initiates the curious phenomenon called “heat precipit,ation” of collagen. In both methods the development of fibrils was followed by measuring optical turbidity; also b0t.h produce fibrils which show the native -640 A spacing in the electron microscope. Gross (1956) and Gross and Kirk (1958) found, using the heat precipitation t,echnique, that, specific elect,rolytes have a marked effect on the rate of fibril formation. Bensusan and Hoyt (1958) came to similar conclusions using procedure (1) to initiate fibril formation. Their results indicated that, electrostatic factors must be operat’ive, but since different ions a t the same ionic st,rength decreased the rate of fibril formation to markedly different extents, the effect is certainly not just one of ionic strength. At constant ionic strength anions exhibited a wider variation than the cations tested. Bensusan and Hoyt, examined the effect, of a a number of cat,ions on the electrophoretic mobility of soluble collagen, and found evidence of specific ion binding. Bensusan and Scanu (1960), in an examination of the effect of iodination of collagen on the rate of fibril formation, found t,hat the rate increased
72
HARRINGTON A N D VON HIPPEL
markedly following iodination and that the only chemical consequence of iodination seemed to be the conversion of tyrosyl residues to the diiodotyrosyl form. These results again implicated the trace amounts of tyrosine found in collagen in the interaction process (see Hodge et al., 1960). As a consequence of these studies, Bensusan and Scanu suggested that the t)yrosyl residue participates in the irit.eraction process in its ionized form, and that the increase in rate on conversion to the diiodo form is due to the decrease in p K which accompanies iodination (for tyrosine, pK =lo; for diiodotyrosine, pK ~ 7 ) They . concluded that the ionization of 8 to 10 tryosyl residues is involved in the activation step; this is in reasonable agreement with their analyt,ical finding of -12 tyrosines per 360,000 molecular weight collagen unit. Ionized lysyl residues also appear to participate in the int,eraction process. The electrostatic basis of the interaction is furt,her supporkd by t.he finding that the rat.e of fibril format.ion increases with decreasing dielectric constant of the medium (Bensusan, 1960) as well as with decreasing ionic strength. Very recent studies by Martin et al. (1961) on the reconstitution of collagen fibrils as a function of pH have suggested that an uncharged imidazole moiety may be crucial in the alignment and binding of adjacent collagen molecules int,o fibrils characterized by the -640 A axial periodicity. Combining t.urhidometric rate measurements with electron microscopic observat,ions, Wood and Keech (1960) have made extensive correlations of fibril dimensioiis wit'h the ionic strength, pH, etc., at] which fibril formation takes place. Their kinetic findings are qualitatively similar tjo those of Bensusari arid Hoyt, (1958), in that fibril formation under most conditions showed a lag pcriod, followed by a sigmoid growth phase. I n a theoretical analysis of the kinetic data, Wood (1960a) showed that the process of fiber format,ion could he divided into a nucleation and a growth phase, corresponding respectively to the observed lag and sigmoid growth periods. Wood (1960b) has also examined the effects of chondroitin sulfat,e and other naturally occurring polyanions on t,he rate of fibril formation; some accelerated fihril formation, while others seemed to inhibit the process. Basically, the data available are in accord with the postulat,e that specifically locat,ed charged groups on one molecule are attracted to specific groups on another, and that most of these groups are probably located in the phosphotungstic acid-staining bands of the collagen fibril. b. Optical Rotatory Properties. In addition to its unique amino acid composition and wide-angle X-ray diffraction pattern, collagen is also chsracterized by unusual optical rotatory properties. X-ray studies have shown that the individual polypeptide chains of collagen in the solid state are coiled into the left-handed, threefold helix characteristic of poly-L-proline I1 (only marginally distorted by the superimposed, right-handed coiled-
STRUCTURE OF COLLAGEN AND GELATIN
73
coil structure). Physieochemical studies have suggested that this type of organization carries through into the collagen molecule. Since it is now well established that the formation of helical structures from random coil precursors is accompanied by a large and characteristic change in optical rotation, it is clear that a structure such as the poly-L-proline I1 helix should exhibit definite and distinctive rotatory properties. And indeed, as we have seen, this is the case. In the last few years, attention has focused primarily on theoretical and cxperimental optical rotatory studies of the a-helix and the @-structuresin proteins and polypeptides (Ee article in this volume by Urnes and Doty). (For recent reviews, see: Schellman and Schellman, 1958,1961; Blout, 1960; Yang, 1961.) Collagen, when considered at all, has been quickly put aside as a rather unpleasant exception to a number of otherwise generally applicable empirical generalizations. Yet the rotatory properties of the poly-Lproline II-type helices of collagen are at least as striking and as characterist,ic as those of the a-helix. A brief comparative consideration of the rotatory parameters of the two systems makes this clear: (1) The rotary dispersion of collagen, like that of most of t,he a-helical proteins, is of the simple type (over the region 400-700 mp) and may be fit,ted to a one-term Drude equation:
where [a]is the specific rotation, X is wavelength, and A and A, are constants characteristic of the system.14 (2) In the denatured (or essentially random coil) form, both collagen (as gelatin) and the a-helix-forming proteins typically exhibit specific rotations close to the mean residue rotation of the component amino acids ([a], N -90" to - 120') and values of A, of -205 to 215 mp. (3) However, in the native form the situation is quite different for the two species. The a-helical proteins generally exhibit a lower specific levorotation ([a], N -20" to -50") and an increased A, (generally to values greater than 230 mp) relative to the denatured form (see the recent and very extensive compilations of Schellman and Schellman, 1961). Collagen, on the other hand, shows a vastly increased specific levorotation ([a],'V -400") and an essentially unchanged A, (see Cohen, 1955; Harrington, 1958; Burge and Hynes, 1959a, b). The optical rotatory parameters for some carefully studied collagens (and the gelatins derived from them by mild heating) are summarized in 14 However, a few proteins with an exceptionally high a-helix content (e.g., some of the myosins) and most of the synthetic polypeptides in the a-helical form, exhibit anomolous dispersion.
74
HARRINGTON A N D VON HIPPEL
Table IX; (further consideration of the rotatory properties of gelatin will be deferred to Section V). Certain ot,hcr features of the optical rotatory propertks :we worthy of comment,. l h t , as mentioned previously and as might be expect,cd on X-ray grounds, the structural portion of the specific rotatioil of collagcn is very close to that obtained with poly-L-proline 11. Thus (see Table IX) a t the sodium D-line the specific rotation of collagen is -400", while the residue rotation is about - 125", yielding a configurational c o n t r i b u t h
-
TABLEIX Optical Rotatory Properties of Collagen and Gelatin
Ichthyocol Calfskin Rat tail Ichthyocol Bovine Codskin Cod swim bladder Herringskin Eelskin Perch swim bladder Ratskin Ichthyocol Dogfish sharkskin
-350" -415" -289 ' -330" -350" -397" -408" -398" -400" -409"
-
-345"
205 217 204 220
218
-110" - 135" -118" -146" - 109.7' -116.0" -124.5' - 125.7" -127.5' -135.0' -124.8' - 122"
205
-
217 217
-
213 -
a
0 c
c C
d d d d d d
e
f
Cohen (1955). Doty and Nishihara (1958). Harrington (1958). Burge and Hynes (1959b). * von Hippel (unpublished). f Lewis and Piez (1961). b
of --275". The [a],for poly-L-proline I1 is more negative (---silo") but the residue rotation of L-proline is also more negative (--250") resulting in a structural rotation, [a10 of about -290" for this material (Harrington and Sela, 1958). Furthermore, A, for poly-L-prolinc I1 is -205 mp; very cose to the value obtained with collagen. These findings suggest, of course, t,hat the polypeptide chain configurations of poly-L-proline I1 and collagen are very similar, but also (unless one is willing t o postulate some rather unlikely cancellation effects) that practically all of the collagen peptide chains in the native molecule are in this configuration. In this connection, it is intcrcsting to note that the specific rotation measured for a number of collagens is essentially constant,
STRUCTURE OF COLLAGEN AND GELATIN
75
while that of the corresponding gelatins varies much more (see Table IX, data of Burge and Hynes). Since the gelatin values reflect primarily the amino acid compositions of the different samples, this also suggests that the specific rotation per residue of the collagen helix does not depend primarily on thc nature of the residues, but is a property of the helix itself (Burge and Hynes, 1959a).
C. The Collagen -+ Gelatin Transition Wc turn iiow t o a more detailed consideration of how the multistranded collage11 molecule is held together and stabilized in the native state, by examining the cverits which take place when this stabilization breaks down during the collagen --+ gelatin transition. 1. Thermal Shrinkage Studies
The collagen -+ gelatin transition can be detected a t various lcvels of structural organization. Thus the so-called thermal shrinkage phenomenon, which has been recognized as a characteristic property of collagen for many years (e.g., see Ewald, 1919) has recently been shown to be a macroscopic manifestation of the molecular collagen -+ gelatin transformation (Garrett and Flory, 1956; Flory and Garrett, 1958). The thermal shrinkage phenomenon may be observed by simply subjecting a bundle of eollagen fibers to slow heating. At a specific temperature, T,, which differs from one species of collagen to another (and may vary slightly with the rate of hcatiiig; see Weir, 1949) these fiber bundles undergo a sharp contraction to less than one-third of their original length. After thermal shrinkage, many of the characteristic properties of the native fiber are lost, including the wide-angle X-ray diffraction pattern (Herzog and Gonell, 1925), the small-angle pattern (Bear, 1944; Wright and Wiederhorn, 1951), arid the resistance to proteolysis by trypsin (Grassmann, 1936). The wideangle X-ray pattern can be at least partially restored by stretching the shrunken fiber to its original length (Astbury and Atkin, 1933) but the small angle pattern is not restored by such treatment (Bear, 1944). Macroscopically, thermal shrinkage is irreversible, though the initial introduction of molecular cross-links between chains (e.g., by subjecting the fibers to mild tanning) does result in a fiber which re-elongates spontaneously when cooled and which retains the small-angle diffraction pattern (Bear, 1942, 1944). Studies of the crystallization of cross-linked polymers have shown that such behavior is not uncommon, and indeed is what one might expect on thermodynamic grounds (e.g., see Mandelkern, 1956). The shrinkage temperature of collagen is very much affected by interaction with various small molecules, including: electrolytes and nonelectrolytes, acids and bases, tanning agents, etc., and much interesting ex-
HARRINGTON AND VON HIPPEL
76
perimental and theoretical work has been done in attempting to uiiderstand these phenomena. Since the work in this area prior to 1956 has been lucidly summarized by Gustavson (1956) we will not attempt to review this material, but will only supplement Gustavson's account by considering certain more recent developments. In particular, two specific areas will be discussed: (1) the correlation between shrinkage temperature and imino acid content; and (2) the demonstration that thermal shrinkage and TABLEX Itelation between Imino Acid Content and Shrinkage and Denaturation Temperatures jor Various Collagens ~
Collagen Calfskin Ratskin Perch swim bladder Pikeekin Ichthyocol Sharkskin Cod swim bladder Codskin Dogfish s h a r h k i n
Pro- Hydroxy- Tqtal linea proline" imino acidsa
138 130 118 129 116 113 103 102 99
94 93 81 70 81 79 57 53 57
232 223 199 199 197 192 160 155 156
T~ 65°C 55°C 55°C 54°C
53°C 40°C
36°C 36°C 31°C 27°C 29°C 29°C 16°C 16°C 16°C
- T ~ j
29°C
-
24°C 28°C 25°C 24°C -
24°C
-
Keferences
d , e, f , 9 g, h
e , h, i d, e g,j
B
e, f, i
k
Residues of amino acid per 1000 total residues. Shrinkage temperature of tissue strips. Denaturation temperature in solution, measured by viscosity or optical rotatiori at pH 3.7. d Doty and Nishihara (1958). Pier and Gross (1960). Gustavson (1955b). Burge and Hynes (1959a). h Gustavson (1956). Esipova (1957). i Eastoe (1957). k Lewis and Pies (1961). a
b
0
the collagen 3 gelatin trarisformatioii are both manifestations of the same molecular process. The latter finding, of course, permits us to carry the pertinent aspects of the fiber shrinkage work over into the molecular domain. a. Shrinkage Temperature and Imino Acid Content. In the course of a study of the thermal shrinkage behavior of a number of skin collagens from different organisms, Gustavson (1953) discovered an interesting correlation between shrinkage temperature and imino acid content. Specifically, he found that, T. seemed to vary directly with the hydroxyproline content (see Table X). Therefore, assuming interchain hydrogen bonds to
STRUCTURE OF COLLAGEN AND GELATIN
77
be primarily responsible for stabilizing the collagen structure, and assuming that these bonds rupture in the shrinkage process, Gustavson (1955b, 1956,1957) postulated that -OH. .O=C- hydrogen bonds, between the hydroxyl group of hydroxyproline and the carbonyl oxygen of the peptide bond, play a key role in the stabilization of collagen, and that it is the rupture of these bonds which precipitates thermal shrinkage. This hypothesis was strengthened by a large number of measurements carried out by Takahashi (Takahashi and Tanaka, 1953; Takahashi and Gustavson, 1956) on the skin collagen of a variety of Japanese teleosts, where again a good correlation between T , and hydroxyproline content was obtained. Other evidence which also apparently favored this point of view has been summarized by Gustavson (1956, 1957).*6 In arriving at this conclusion, Gustavson dismissed the apparently equally good correlation between either proline or total imino acid content and T , (see Table X) on the grounds that proline cannot participate as a hydrogen donor in hydrogen bonding. Thus Gustavson felt that the presence of proline would, if anything, reduce rather than increase the stability of collagen. Somewhat later, on the basis of other evidence, it became apparent that hydrogen bonding might not be the sole factor involved in stabilizing collagen. (1) Szent-Gyorgyi and Cohen (1957) suggested that the presence of large amounts of proline (or hydroxyproline), which cannot be accommodated in an a-helical structure, might predispose a polypeptide chain to take up the alternative poly-L-proline II-type configuration. (2) Harrington and Sela (1958) demonstrated that poly-L-proline exists in solution as a single chain structure cast into the poly-L-proline I1 configuration (see Section 11) indicating that this structure is stabilized by restrictions to the rotation of the pyrrolidine ring about the peptide backbone. Harrington (1958) also suggested that the presence of contiguous pyrrolidine ring-containing residues might affect the geometry of the polypeptide chain in proteins (particularly collagen) by similar mechanisms. (3) In the course of an examination of the gelatin 4 collagen-fold transition, von Hippel and Harrington (1959) proposed that the poly-L-proline II-type configuration develops along single gelatin chains as an intermediate step in the formation of the collagen-type structure. This suggested quite specifically that restricted rotation about both the lfi In particular, acetylation experiments seemed to lend strong support. Gustavson (1954) found that complete N-acetylation of bovine collagen does not affect the thermal shrinkage temperature of the fibers. On the other hand, combined N - and O-acetylation (blocking both amino and hydroxy groups) lowered T, by approximately 20°C.
78
HARRINGTON AND VON HIPPEL
c,-c
4
0
Bond (ii)
and the peptide bonds adjacent to pyrrolidine rings is involved in stabilizing the collagen-fold (see Section V). (4)Burge and Hynes (1959a) compared the proline and hydroxyproline content with the dilute solution denaturation temperature (see below) of several collagens, and again found good correlation between proline, hydrxyproline, and/or total imino acid content and T , (see Table X), t,hough the correlation with total imino acid content secmed the best of the three. (5) Piez and Gross (1960) reported very careful measurements of tho amino acid composition of a great many collagens, and compared these results with values of T , culled from the literature (see Tahle X). They also found statistically significant correlations between T , and proline, hydroxyproline, and t,ot,al imino acid content; the best correlation again being that betwecn T , and total imino acid residues (see Piez, 1960). These observations led Burge and Hynes, arid Piez and Gross to suggest that the correlation between T 8 (and T,) and total pyrrolidine content is the significant one. This conclusion, in conjunction with the considerations cited above, suggests that, the stereochemical properties of pyrrolidine ringcontaining residues in the polypeptide chain environment, rather than interchain hydrogen bonding, might be the key factor in stabilizing the collagen structure. b. Relation between the Thermal Shrinkage of Fibers and the Dilute Solution Transition. Both the macroscopic thermal shrinkage of bundles of collagenous tissue, and t,he t>hermaldenaturation of the soluble collageu derived from such t,issue, have been known and studied for many years. However, unt,il fairly recently the connection between these two phenomena has not been entirely clear. About *5 years ago the feeling became relatively general that these two thermally induced alterations must both be manifestatioiis of a hasically similar process, presumably involving thc collapse of the rigid three-stranded collagen molecule. I n this view, fiber shrinkage occurred at higher temperatures than the dilute solution transit,ion because, in the solid state, both the stabilization provided by the crystallization energy (the energy of interaction of the native collagen molecules) and the intramolecu1a.r forces stabilizing the molecules themselves, must be overcome (Boedtker and Doty, 1956). To investigate this idea, Esipova (1957) and Doty and Nishihara (1958) examined the temperatme at which solutions of various types of collagen were transformed to gelatin, and compared these measurements of T a with thermal shrinkage temperatures obtained by Gustavson and others.
STRUCTURE OF COLLAGEN AND GELATIN
79
They found that in all classes for which data were available, the temperature differences ( T , - To)were essentially constant a t 27” f 3°C. This strongly suggested that differences in thermal stability of the various collagens depend on intramolecular processes rather than intermolecular interactions. And this conclusion, in turn, seemed to make possible a clear choice between collagen structures I and 11. One of the major differences between structures I and I1 (see Table VII) lies in the orientation of the hydroxyl group of hydroxyproline relative t,o the rest of the molecule. In structure I, this group can form hydrogen bonds wit,h reccptor groups within the same three-stranded collagen molecule, while in structure I1 these OH groups are oriented away from t8hemolecule mid can only part,icipate in int,ermolecular hydrogen bonding. Thus, accepting Gustavson’s explanation for the correlation between hydroxyproline and T, , Esipova, and Doty and Nishihara, concluded that their results provided substantial support for the collagen I structure since differences in hydroxyproline content only seemed to affect the magnitude of the intramolecular stabilization. On the other hand, the more recent views on the role of the imino acids in stabilizing the collagen structure undercut the basis of this argument (Burge and Hynes, 1959a). In their treatment of this problem, Garrett and Flory (1956; Flory and Garrett, 1958) accepted the point of view of some of the earlier workers (e.g., Wohlisch, 1932; Kuntzel, 1937) that the thermal shrinkage process could be treated as the “melting” of crystalline arrangements of polypeptide chains, and suggested, in agreement, with the view advanced by Boedtker and Doty (1956), that the dilute solution transition represents this melting process stripped of its intermolecular component. Flory and Garrett made a very careful study of the melting of dried samples of bovine achilles tendon and rat tail tendon as a function of collagen concentration, utilizing measurements on samplcs with collagen volume fractions, v 2 , in et>hyleiwglycol ranging from 0.84 to 0.0004. Some of their results, plotted as melting temperature versus v 2 , are shown in Fig. 16. The measurements at the higher concentrations were made using either dilatometry or direct, observation with a polarizing microscope.1G At the lowest concentratioii (point 10 in Fig. 16) viscometry was used to determine the melting temperature. As Fig. 16 shows, the transition temperature varies monotonically with volume fraction over the entire range, and the experimental pointjs fall almost exactly on the theoretical curve calculated by substituting the parameters characteristic of collage11 into the usual polymer melting point, c1quat.ion. (See Flory, 1953; Mandelkerii, 1956; Flory, 1961.) These l6 In the dilatometric measurements, the temperature of the latent volume change due to fusion was measured, while melting evidenced itself in the polarizing microscope as the disappearance of the depolarization due to the birefringence of collagen fibers.
80
HARRINGTON AND VON HIPPEL
results, when combined with a demonstration of the reversibility of this transition, proved conclusively that both thermal shrinkage in the solid state and the collagen ---f gelatin transformation in dilute solution are manifestations of a first-order crystalline -+ amorphous phase transition. (Studies of the reverse process in dilute solution, the gelatin 4 collagen transition, are treated in detail in Section V.)
q. FIG.16. Melting temperature plotted as a function of composition for collagenethylene glycol mixtures: Solid curve is calculated on the basis of a normal polymer melting relation (see Flory, 1953). (From Flory and Garrett, 1958. Reproduced with kind permission of the American Chemical Society.)
2. Dilute Solution Studies of the Collagen -+ Gelatin Transition
As mentioned previously, a rather dramatic alteration in physicochcmical properties takes place over a narrow temperature interval when a solution of soluble collagen is subjected to gradual heating. This process may be conveniently observed using viscometry or optical rotation, since both the specific viscosity and the specific rotation change enormously as a consequence of the collagen -+ gelatin transition. Thus the intrinsic viscosity [q] falls from the 12-15 dl/gm characteristic of soluble collagen (see Table VIII) to about 0.4 dl/gm., while the specific rotation [a],changes from approximately -400" to --125" (Table IX). Figure 17 shows typical phase transition profiles for three different collagens monitored visco-
STRUCTURE OF COLLAGEN AND GELATIN
81
metrically, while Fig. 18 demonstrates that the changes observed viscometrically are equivalent to those measured using optical rotation. Examination of Fig. 17 reveals that the temperature interval over which the transition takes place has a finite breadth, and thus may be characterized by various temperatures defined in different ways. T o , the temperature quoted in Table X, is defined as the temperature of the midpoint of the transition. If one assumes: (1) that the fractional change in intrinsic viscosity (or specific rotation) is proportional to the fractional conversion of collagen from a single completely native state to a single completely denatured state, and (2) that equilibrium between these two forms is attained at every temperature, then the relation T o = AH/AS applies and these thermodynamic parameters can be calculated from data such as
TEMPERATURE OF 30 MINUTE HEATING ("GI
FIG.17. The collagen -+ gelatin transition for various collagens, measured viscometrically. (From Doty and Nishihara, 1958.)
that of Fig. 17. This has been done by Boedtker and Doty (1956) for ichthyocol, and by Burge and Hynes (1959a) for various other collagens. However, subsequent measurement of the rate at which equilibrium is attained at temperatures in the transition region (Harrington and von Hippel, 1961) showed that times up to 24 hr may be required to reach the final value at a given point. Since Boedtker and Doty, and Burge and Hynes constructed their transition curves by waiting only 30 min at each temperature, complete equilibrium at intermediate temperatures may not have been attained. This leads to artificially sharpened transitions and elevated values of T , . This difficulty, coupled with the possibility that condition (1) above may also not apply (see Flory and Weaver, 1960) suggests that calculations of AH and A S made in this way should not be too rigorously interpreted. These considerations also indicate that probably tjhe more significant parameter for characterizing phase transitions of this sort is TW,, defined as the temperature at which the most ordered seg-
82
HARRINGTON AND VON HIPPEL
ments of the crystalline structure melt (see voii Hippel and Harrington, 1960), although this temperature is often more difficult to measure accurately. The temperature at which a givcn soluble collagen undergoes the collagen -+ gelatin conversion (defined either as T , or T,,J is a usoful parameter for identification and characterization purposes. However, the transition temperature is constant only when measured in relatively dilute salt
3
TIME (MINUTESt
FIG-.18. Comparison between the rate of the collagen -+ gelatin transition for soluble calfskin collagen a t 35.9"C, measured as the fractionaI change in specific viscosity (solid line) and specific rotation (dotted line). (From Doty and Nishihar:t, 1958.)
solutions. Concentrated solutions of neutral salts, such as LiBr and KCNS, have a marked depressant effect on TD (e.g., see Bocdtker and Doty, 1956; Harrington, 1958). Competitive hydrogen-bonding agents, such as concentrated solutions of urea and guanidine-HCl, also lower the transition temperature markedly (see von Hippel and Harrington, 1960); T n also is lowered slightly a t pH values close to 1 (Burge and Hynes, 1959a).I7 The mechanisms whereby some of these diverse agents exert their effects 17 The extensive and presumably closely related studies on the effects of various electrolytes, urea, and pH on the thermal shrinkage of collagen in the solid state have been comprehensively reviewed by Gustavson (1956).
8TltUCTURE OF COLLAGEN AND GELATIN
83
on collagen arid gelatin will be discussed in Section V. Anticipating this discussion, it appears that the neutral salts may exert their effects through a modification of solvent-polypeptide chain interaction, though binding of ions to polar groups may also play a role. Urea and guanidine-HCI doubtless rupture hydrogen bonds, perhaps between water and carbonyl oxygen groups (von Hippel and Harrington, 1960; Harrington and von Hippel, 1961). Low pH probably lowers T D by increasing intramolecular electrostatic repulsion (Burge and Hynes, 1959a).
D. The Use of Proteolytic Enzymes in Structural Studies o j Collagen l’rotcolytic enzymes have been used extensively in the study of protein structure. First, of course, purified proteases have been employed to catalyze the cleavage of specific peptide bonds in proteins in the processof establishing amino acid sequences and distributions. Within the last few years it has also become apparent that proteolytic enzymes can be utilized to obtain a t least semiquantitative information about protein (and nucleic acid) configuration (or secondary-tertiary structure) as well as about amino acid sequence. This use of proteases can be divided into two general areas: (1) Physicochemical examination of the changes in protein (substrate) structure which accompany progressive proteolytic degradation: here the enzyme is used to rupture specific bonds a t known rates, and the molecular consequences of this action are studied. (2) Analysis of the kinetics of proteolysis of a protein substrate as a measure of steric or configurational features in the vicinity of suceptible pept.ide bonds: in t,his approach the enzyme molecule itself is used as a “configurational probe” to examine local aspects of substrate structure. Both approaches, if properly applied in favorable situations, seem capaable of providing molecular insight of a different sort from that obtained via the usual physicochemical methods, and both will be discussed below as they have been applied in the study of collagen. 1. Physicochemical Studies of Collagen during Proteolysis
This approach, monitoring the changes in a macromolecular substrate as a function of the number of enzymatic breaks in the polypeptide (or polynucleotide) backbone, was first employed by Thomas (1956) and Schumaker et al. (1956) to prove that the native deoxyribonucleic acid (DNA) molecule is composed of two chains, and to measure the strength and distribution of the interchain hydrogen bonds which hold the two chains together. I n these studies, Thomas related changes in viscosity and light scattering to the number of enzymatic breaks, while Schumaker et al. focused primarily on the viscometric changes. More recently, Sinsheimer (1959) has used a similar procedure to show that the DNA isolated from
84
HARRINGTON AND VON HIPPEL
the 6x174 virus is single-stranded. This technique has been applied to soluble collagen by von Hippel et al. (1960), who followed the changes in specific viscosity, light-scattering molecular weights and radii of gyration, nondialyzable protein and optical rotation which accompany the degradation of native ichthyocol by the collagenase isolated from Clostridium histo1yticum. Von Hippel et al. found by light scattering that neither the molecular weight nor the radius of gyration of the ichthyocol molecule changed markedly during collagenolytic degradation at 5°C. Moreover, though an,the molecular weight per free N-terminal residue, eventuslly fell to -500, very little of the protein became dialyzable if both the enzymatic treatent and the dialysis were conducted at low temperature. (On the other hand, most of the protein did become very rapidly dialyzable if dialysis was conducted a t temperatures above T D.) These results are consistent with the current view of collagen as a three-stranded molecule held together by interchain hydrogen bonds, and permitted a crude estimate of the number of intact interchain hydrogen bonds needed to hold a cleaved peptide fragment to the rest of the molecule a t low temperatures. Apparently less than ten are required.18 Concurrent optical rotatory studies showed that the molecule not only retains most of its integrity in terms of mass, but also that the intact interchain hydrogen bonds largely maintain the helical configuration of collagen despite very extensive backbone cleavage (see Fig. 19). These results were contrasted with viscometric studies, which showed that the specific viscosity of soluble ichthyocol collagen decreases linearly and without an initial lag period when plotted as a function of p , thc probability that a given susceptible peptide bond has been split. If one assumes (see Schumaker et al.) that: (1) the substrate is a rigid n-stranded polymer held together by relatively weak interchain links; (2) the enzyme attacks susceptible bonds of single strands a t random; (3) changes in the specific viscosity are detected only when the molecule is split into two smaller n-stranded pieces by cleavage of all the chains at loci which arc sufficiently close so that the intervening interchain links cannot hold the chains together; then the following equation applies: where (vSp,t/Tlsp .o) is the ratio of the specific viscosity at time t to that at time zero, n is the number of strands in the molecule, p is the probability l* It is interesting to note, in this connection, that Thomas, and Schumaker et al. found that only a short sequence of intact interchain hydrogen bonds (of the order of 3-5)seemed to be needed to prevent the dissociation of the two chains of DNA a t room temperature.
STRUCTURE OF COLLAGEN AND GELATIN
85
of bond cleavage defined above, and K is an arbitrary constant. Under these conditions, it is clear that qep can be a linear function of p only if n = 1; that is, if the efficiency of an enzymatic break with respect to its effect on qBpis a constant from the start of the reaction. Yet this result is
143 ‘C
20xt -
xo
10 -
08 06 -
04
-
I 20 40 60 80 100 I20 140
01
0
TIME (MIN )
FIG.19. Comparison of the fractional decrease in specific viscosity and specific rotation as a function of time after adding collagenase to soluble ichthyocol collagen at 14.3”C. (From von Hippel et al., 1960. Reproduced with kind permission of the American Chemical Society.)
incompatible with the three-chain collagen molecule and the observed insensitivity of the particle mass to enzymatic attack, unless assumption (3) is invalid and each backbone cleavage increases the molecular flexibility (and thus decreases qap) regardless of whether the molecule separates into two parts or not. These findings suggest that the rigidity of the collagen molecule depends upon the “intactness” of all three polypeptide chains, and that the super-helix, because of its extremely large pitch, does not con-
86
HARRINGTON AND VON HIPPEL
tribute very much to the molecular rigidity. These results also clarify the basis of the viscometric assay for collagenase introduced by Gallop et aE. (1957b), in which log qap is plotted against time for soluble ichthyocol in the presence of collagenase, and the slope of the resulting straight line is related directly to the activity of the enzyme preparation. The progressive decrease in the specific viscosity of a solution of ichthyocol subjected to collagenolytic attack is compared to the accompanying fall in specific rotation in Fig. 19. This shows strikingly, as pointed out above, that the increase in molecular flexibility due to enzymatic cleavage is not accompanied by an equivalent destruction of the compound collagen helix. It is interesting to contrast this plot with Fig. 18, which demonstrates that the specific viscosit,y and the rotation fall together when the rigid, three-stranded collagen molecule is converted to random-coil gelatin.
2. Proteolytic Enzymes as “Probes” of Collagen Structure The use of proteolytic enzymes as “configurational probes” grows out, of the very old observation that, a native protein is often partially or even completely resistant to prot>eolysisby an enzyme which readily attacks the denatured form (e.g., see Linderst>r@m-Lang, 1952). Since denaturation, by definition, does not alter covalent bonding or amino acid sequence, configurational (steric) factors must &her prevent the enzyme from reaching the susceptible bond in the native molecule, or prevent it from orienting over the susceptible bond in such a way as to successfully catalyze cleavage. Such behavior was observed by Grassmann (1936), for solid collagen fibers with respect to tryptic hydrolysis; trypsin would not attack native collagen fibers but easily degraded thermally shrunken specimens. The analogous observation in solution was made by Gallop et al. (1957a) who showed that native soluble collagen is not attacked by trypsin, but that after conversion to gelatin it is readily digested. These qualitative findings suggested that analysis of the kinetics of proteolysis might yield quantitative data on polypeptide chain configurations in certain favorable cases. Studies of the tryptic hydrolysis of myosin and of the collagenolyt,ic degradation of collagen seemed to confirm this view, and led Harringt~on et al. (1959) to propose the use of proteolytic enzymes as probes of the secondary structure of fibrous proteins, specifically as a measure of the crystalline and amorphous (in an X-ray diffraction sense) regions along the polypeptide chains. Subsequent work has supported this crystallineamorphous interpretation for the myosin-trypsin study, though in the collagen-gelatin-collagenase system the situation appears to be more complex. Mihalyi and Harrington (1959) found that the digestion of rabbit myo-
STRUCTURE O F COLLAGEN AND GELATIN
87
sin, followed by pH-stat methods, did not obey kinetics of an integral order. However, further investigation revealed that the kinetics could be fitted nicely by assuming that the over-all reaction consists of two independent apparent first-order reactions proceedings] a t markedly differing rates.lg An investigation of the kinetics of proteolysis of ichthyocol collagen (von Hippel et aE., 1960) and cold ichthyocol gelatin (von Hippel and Harrington, 1959) by collagenase revealed similar behavior; in each case the kinetics could be reduced to the sum of two concurrent apparent first-order reactions. A typical example of the type of data obtained with thc collagen-collagrnase system, illustrating the analysis into two reactioils, is presented in Fig. 20a, where the fraction of the total bonds cleaved is plotted as a function of time. Extrapolation of each linear segment back to the ordinate gives the fraction of the total bonds split in that reaction, and the apparent first-order rate constant for each reaction can be derived from the slopes of the corresponding lines. These findings support the view that the collagenase-sensitive (or trypsin-sensitive, in the myosin-trypsin system) peptide bonds of ichthyocol collagen and cold gelatin can be divided into two general reaction classes in terms of their susceptibility to enzymatic attack. Reactions run at elevated trmperatures demonstrated that the differences between the two classes of suseeptiblr bonds are primarily configurational rather than chemical, since heating myosin to 41°C resulted in the transfer of some of the trypsin-sensitive peptide bonds from the slower to the faster reaction class (Harrington et at., 1959). Even more dramatic was the finding that heating collagen to temperatures above the collagen -+ gelatin transition temperature (27°C for ichthyocol) resulted in all the bonds being split in a single apparent first-order reaction (Fig. 20b) differing in terms of thermodynamic parameters from either of the reactions observed with native collagen (see Table XI). This seemed to imply that the differences between the susceptible bonds, at least in the collagen-collagcnase-gelatin system, are entirely configurational in origin. I n Fig. 21, the logarithms of the apparent first-order rate constants obtained from a series of pH-stat runs on ichthyocol collagen a t various temperatures are plotted against the reciprocal of the absolute temperature as an Arrhenius plot. Clearly the data fall onto three straight lines: those numbered (2) and (3) for the two reactions a t temperatures below T , , line (1) for the single reaction at temperatures above T D. The apparent enthalpy ( A H * ) , free energy (AF*), and entropy (As*) of activation, together with the fraction of the susceptible bonds split in each reaction, are compiled in Table XI. Similar results were obtained by Connell (19GO) in an examination of the kinetics of tryptie digestion of cod myosin.
88
HARRINGTON AND VON HIPPEL
Examination of Table XI and Fig. 21 brings out several interesting points: (1) There is a sharp break in the Arrhenius plot a t the collagen -+ gelatin conversion temperature; this is also true in the reverse reaction (see Section V). COLLAGENASE ON COLLAGEN pH 8 0 1955 C '
a
-
pm-pt $03
I I
TIME (MINI
Fro. 20a. Fraction of the susceptible bonds of soluble ichthyocol collagen cleaved by collagenase, as a function of time at 19.5OC. (a), Experimental pointjs; (e),fast reaction (calculated by subtracting the extrapolated slow reaction from the experimental data. (From von Hippel et al., 1960.)
(2) Above T D, all the collagenase-susceptible bonds appear to be split in a single apparent first-order reaction, with AH* and AS* (-15 kcal/ mole and -0 e.u., respectively) close to the values usually associated with the proteolysis of denatured proteins and synthetic substrates. (3) The corresponding parameters for the two reactions in collagen and cold gelatin are much larger than those for the reaction above T , . (4) Approximately the same distribution of susceptible bonds between
89
STRUCTURE OF COLLAGEN AND GELATIN
the fast and the slow reaction obtains at all temperatures in both native collagen and cold gelatin. Points (1) and (2) above suggest, in agreement with physicochemical evidence, that above T, gelatin exists as an open, essentially random coil structure with the susceptible peptide bonds optimally available to the COLLAGENASE ON GELATIN
1.0
0.9 00
0.7 06
b
0.5
04
b-Pt pal
0.3
0.2
01
I
I
1
I
I
I
20
30
L
10
40
50
60
70
TIME
(MIN.)
FIG.206. Fraction of susceptible bonds of ichthyocol gelatin cleaved by collsgenase, as a function of time at 37.35"C. (From von Hippel and Harrington, 1959.)
enzyme. Below T D , the bonds in both collagen and gelatin (which undergoes a reversion to the collagen-type structure, see Section V) are not as available to the enzyme. Hence we may speculate that since collagenase is a highly specific enzyme (catalyzing only the cleavage of the sequence Z.Pro.X.Gly.Pr0.Y) t,hat the rate of catalysis may be strongly dependent on the orientation of the two required penultimate pyrrolidine rings relative to the polypeptide chain. Above T o , rotation of the pyrrolidine rings
90
HAllHINGTON AND VON HIPPYL
about the polypeptide chain should be relatively unhindered, and the most favorable orientation for catalysis easily attained. However below T o , where the chains as a whole exist in the poly-L-proline 11-type conTABLEX I TherrrLodynaniic Data, Collagenase on Ichlhyocol Collagen and Gelatin"
% ' (T) !?-
Substrate _-
Collagen (fast reaction) Collagen (slow reaction) Gelatin Gelatin (fast reaction) Gelatin (slow reaction) 0
10-23 10-23 28-37 10-25 10-25
AH*
(i%$ (kcal/ mole) mole)
Bonds
(%)
AF*
(kcal/ mole) -
10 f 3 84 f 4 100 18 f 4 82 f 4
1-42 +47 +15 +23 $30
$41 +46 $14 +22 +29
$15 $15 $14 +14 +15
+90 4-110 $1 $30 $51
f 5 f6 f7 f4 f4
Data from von Hippel and Harrington (1959) arid von Hippel et al. (19GO).
COLLAGENASE ON COLLAGEN
Oo0t
I
3.20
3.30
3 40
3.50
3 60
+(x1031
FIG.21. Arrhenius plots for the proteolysis of ichthyocol collagen arid gelati11 by collagenase. pH-stat data: (1) gelatin above T D ; (2) collagen below T D , fast reaction; (3) collagen below T D , slow reaction. (From von Hippel et al., 1960.)
figuration, the rate of catalysis might depend strongly on the relative orientation of the pyrrolidine rings and 011 the nature of residue X. We may assume that the enzyme distributes the susceptible bonds into the two observcd reaction classes 011 the basis of some setluence-determiiicd configurational basis (see Section V). Then the uniformly higher AH*
STRUCTURE OF COLLAGEN AND GELATIN
91
+
(about +20 kcal/mole) and AS* (about 60 e.u.) found for each class in native collagen relative to cold gelatin, implies that these differences may constitute a measure of the “tighter” folding of the polypeptide chains in the collagen molecule superimposed on the effect of local configuration differences.20
E. The Role of Water in the Collagen Structure Several types of evidence suggest that water is intimately involved in t,he struct>ureand stabilization of collagen. For example, Gustavson (1956), in reviewing the thermal shrinkage phenomenon, emphasized that the substitution of other solvents for water markedly influences the shrinkage temperature and other properties of the fiber. The molecular consequences of the hydration of collagen have been most extensively and revealingly st,udied by X-ray diffraction techniques. It has been known for many years that the characteristic wide-angle X-ray pattern of collagen or cold gelakin is largely destroyed b y dehydrat,ion, and at least partially restored by rewetting the material. This suggest,ed that water might be involved in stabilizing the collagen structure, and led Rougvie and Bear (1953) to undertake a quantitative study of the wrious features of both the wide- and small-angle diffraction patterns of colla,gen (kangaroo tail tendon) which are affected by moisture content. Rougvie and Bear constructed a moisture sorption isotherm (Fig. 22) and found that the primary sorption amounted to -13.0 gm of water per 100 gm of collagen when analyzed by the standard B.E.T. method (Brunauer et al., 1938). Correlating moisture sorpt,ion with X-ray changes, they found that the hydration-sensitive equatorial spacing observed in t,he wideangle X-ray pattern shifted from -10.6 A for dry fibers to a maximum value of 14.6 A in completely wet specimens, and that the small-angle meridional macro-period increased from approximately 600 A to -670 A with increasing hydration. Accompanying changes in line intensities in the small-angle pattern also indicated that improved ordering accompanied the increase in moisture content (see Fig. 14). Rougvie and Bear concluded t,hat the total moisture sorption range could be divided into four successive intervals (in order of increasing water content) : (1) Hydration of the polar side chains of the residues located a t the disordered fibrillar band regions. (2) Hydration of polar groups in the interbands, accompanied by some * O Very recently, Orekhovich e t al. (1960) have also reported marked differences between the rate a t which collagen is degraded by collagenase in the native and in the denatured state. They feel t h a t their results support t h e “local configurational changes involving proline residues” interpretation of these phenomena offered by von Hippel and Harrington (1959).
92
HARRINOTON AND VON HIPPEL
lateral chain separation as evidenced by the increase in wide-angle equatorial spacing. (3) Straightening of kinked chains and further lateral separation. (4) The attainment of apparently complete lateral separation of the chains (or chain bundles, see below) at both the bands and interbands. Several years later, Borge et al. (1958) reconsidered some of the hydration data of Rougvie and Bear from the point of view of the stereochemis-
Relative
humidlty (%)
FIQ.22. Moisture sorption isotherm (25°C) for kangaroo tail tendon. The locrttions a, b, c, d, and e divide the data into the four successive intervals discussed in the text. (From Rougvie and Bertr, 1953.)
try of the collagen I1 structure. They pointed out that since only one-third of the carbonyl oxygens and peptide nitrogens of this structure are involved in hydrogen bonding within the three-chain collagen unit, a considerable number of polar groups are left unbonded. Specifically, they noted that if one assigns one water molecule t o each of the unbonded peptide carbonyl and amide nitrogen groups, plus one to each polar side chain, a total of 19.7 gm of water per 100 gm of protein should be taken up in the primary reaction, compared to the 13.0 gm measured by Rougvie and Bear. However, they pointed out that this experimental value is
STRUCTURE OF COLLAGEN AND GELATIN
93
close to that calculated for one water molecule per polar side chain plus one for every two unbonded carbonyl groups (13.3 gm). Examination of the three-chain collagen I1 model (incorporating either the Gly.Pro.Hypro or the G1y.Pro.X repeat) showed that water molecules could be systematically placed so as to form hydrogen bonds with two carbonyl oxygens simultaneously in at least two ways: (1) by bridging the carbonyl oxygen of the hydroxyprolyl (or X) residue and that of the adjacent glycyl residue on the Same chain; or (2) by bridging the glycyl carbonyl oxygen and that of the adjacent hydroxyprolyl (or X) residue of the next chain in the clockwise direction (viewed from the 6-terminal end of the collagen I1 model). Burge et al. felt that neither arrangement was entirely stereochemically satisfactory, and therefore did not pursue these possibilities, but concentrated their attention on the effects of singly-bonded water on the collagen wide-angle pattern.2l To this end, Bradbury et al. (1958) calculated the diffraction pattern expected from collagen I1 with water molecules singly bonded in every possible systematic position along the chains. This amounted to approximately 25 gm of water per 100 gm of collagen. The patterns calculated on this basis were compared to those obtained experimentally, and the agreement seemed somewhat better than that between the experimental results and the transforms calculated for the anhydrous collagen I1 structure, though the agreement was not good enough to definitely exclude other hydration arrangements. It did seem clear, however, that systematically disposed water molecules constitute an important portion of the diffracting structure. An X-ray study of collagen hydration has also been carried out recently by Esipova et al. (1958). These workers measured moisture sorption isotherms and attempted to relate the results to intensity changes in the wide-angle diffraction pattern, in order to establish the position of the absorbed water with respect to the diffracting portions of the collagen fiber. They also found that hydration increased the crystallinity of the collagen fiber. More specifically they inferred that the oxygen atoms of the water bound to the ordered portions of the structure seemed to lie very close to the axis of the polypeptide chains (within -3 A) and to be arranged in a semiregular fashion along the chains (-3 A apart). Moreover, the stoichiometry seemed to suggest that nonhydrogen-bonded peptide carbonyl groups were primarily involved. On the basis of these results and the stereochemical findings of Burge et al., Esipova and co-workers suggested that the crystalline portions of the collagen structure might be sta.bilized by 21 Recent comparative studies on the rate of formation of the collagen-fold in H20 and D20 seem to support the possibility of intrachain water bridges linking adjacent carbonyl oxygens. These studies are discussed in Section V.
94
HAHBINGTON AND VON HIPPEL
doubly hydrogcri-bonded water bridges of the type :
giving rise to continuous chains of structurally incorporated water along the fiber axis in the diffracting regions. Similarly hydrated structures have been proposed for poly-(glycyl-L-proline) by Millionova and Andreeva (1958). Infrared measurements have provided additional information about hydration arid hydrogen bonding in collagen. Specifically, Bradbury et al. (1958) examined the rate of deuteration of films of native ratskin collagen, and found three groups of labile protons differing markedly in ratc of exchange. The most rapidly exchangeable group was attributed to partially degraded portions of the specimen, the second group t o labile protons on side chains and N-H groups not involved in N-H. . .O=C-- hydrogenbonding, and the vcry slowly exchanging protons were assigned to N-H groups involved in interchain hydrogen bonding. A value of 1:1.3 was determined for the ratio of the number of N-H groups involved in interchain hydrogen bonding to those bonded directly to water, in reasonable accord with expectations based on the collagen I1 model. All groups showed essentially instantaneous deuteration in heat-denatured (gelatinized) specimens. Bradbury and co-workers also employed infrared techniques to obtain a moisture sorption isotherm for ratskin collagen. Their results are very similar to those obtained by ltougvie and Bear for kangaroo tail tendon (Fig. 22) ; however, they attributed the lateral separation accompanying increased hydration to the separation of adjacerit three-stranded collagen units, rather than to the separation of individual chains as suggested by Rougvie and Bear. Bradbury and co-workers also concluded that water plays a major role in the stabilization of both the intermolecular and the intramolecular collagen structure. Another investigation of hydrated collagen has been conducted by F’raser and MacRae (1959) who concluded on the basis of infrared dichroic mmsurements that the bound water molecules are primarily singly bonded to the -C=O groups which project radially outward from the collagen molecules. The bound water molecules seemed to be preferentially oriented normal to the fiber axis. These studies all point to water as an important, indeed, an integral, component of the collagen structure. It is expected that future work will establish unequivocally the precise arrangement of the water molecules within the collagen framework.
STRUCTURE O F COLLAGEN AND GELATIN
V. THE STRUCTURE OF GELATIN The dissociation of the polypeptide chains of collagen by thermal or chemical proeessrs leads to products variously termed gelatin. In thr present section we propose to consider certain of the physicochemical properties of gelatin, looking toward a deeper understanding of the structure and chemistry of collagen. For many years gelatin has been utilized in a large number of industries, and thus for economic reasons the procedures used for extraction have been based on the attainment of maximum yield. Since it has been of great interest, from the industrial point of view, to learn more about the properties of these gelatins, the majority of the published studies in this area have been carried out on such samples. Because of the diverse and rather rigorous nature of the extraction procedures it is not surprising that a rather broad spectrum of molecular properties has been reported. On the other hand, it will be apparent from the discussion in Section IV that even the mildest gelatinization procedures may not lead to a system of completely uniform polypeptide chains. Evidence is accumulating which suggests that the component chains of the intact, fundamental collagen unit may not be identical, either with respect to size or amino acid sequence. Ideally, then, before undertaking detailed studies, the subunit chains should be dissociated by mild procedures and resolved either chromatographically or by some equivalent analytical technique. Undoubtedly, this approach will be used a great deal in future studies and we may confidently expect fruitful results. However, despite the expected heterogeneity, studies of gelatin systems have made possible a much deeper insight into the molecular properties of collagen and, indeed, have made substantial contributions to other areas of polymer and protein chemistry. Two processes are commonly used in the commercial preparation of gelatin. In the first process, hide or demineralized bone is extracted over a prolonged period of time in alkaline solution. During this steeping operation hydrolytic changes occur, leading to a release of collagenous material which is subsequently gelatinized at neutral pH a t temperatures of 60"65°C. In the alkaline medium used for extraction, the amide groups of the glutamine and asparagine residues are released, resulting in a gelatin with an isoelectric point at about p H 4.9-5.0 (Bowes and Kenton, 1948; Ames, 1952; Kenchington and Ward, 1954). Additionally, a significant number of peptide linkages are split in the alkali pretreatment, as demonstrated by the appearance of N-terminal residues (Bowes and Moss, 1953; Deasy, 1958; Courts, 1954, 1958, 1960). The second process involves soaking skin, bone, or tendon in dilute acid,
96
HARRINQTON AND VON HIPPEL
followed by extraction with warm water at an acid pH. In this instance the gelatin is not deamidated (Ames, 1957; Kuntzel et al., 1958; Veis et al., 1958; Courts, 1960), and an isoelectric point of about pH 9 is obtained for the resulting product. Generally speaking, acid-extracted gelatin exhibits fewer N-terminal residues per unit weight than does alkali-processed gelatin, but it is clear from the work of Courts (1960) that some degradation of the gelatin chains occurs during the steeping operation. Gelatins may also be derived from soluble collagen preparations by treatment with urea, sodium thiocyanate, lithium bromide, or by heating to temperatures above T D . Since these collagen molecules have a very uniform size distribution, it is to be expected that the derived gelatiiis would exhibit the most meaningful physicochemical propertips. From the pure protein-chemical point of view, it is a pity that so many of the investigations of gelatin have been carried out using the relatively degraded acid- or base-processed material.
A . The Molecular Properties of Gelatin at Rocm Temperature and Above 1. Size and Shape a. Gelatins Derived from Insoluble Collagens. In 1944, Scatchard et al. published a short report on the molecular properties of a soluble, commercial, alkali-processed gelatin derived from bone collagen. In this study it was assumed that collagen consists of long polypeptide chains, and that in the preparation of gelatin the bonds along each chain are hydrolyzed a t random a t about the same rate. A few bonds, equally spaced along the chain, were assumed to be hydrolyzed much more rapidly. According to this view, there should exist an ideal parent gelatin molecule, defined as the chain segment between two of the easily hydrolyzable bonds. Coupling tho number-average molecular weight a,, (osmotic pressure) and weight-aver(sedimentation equilibrium)) and using the age molecular weight, Montroll-Simha theory (1940) to predict the size distribution to be expected from random degradation, Scatchard et al. (1944) estimated the molecular weight of the parent gelatin chain to be about 110,000. In a more recent, (light scattering) very thorough study, Boedtker and Doty (1954) found of a similarly prepared gelatin (Knox, P20) to be 97,000. Calculation also revealed the root-mean-square end-to-end distance (P)l/* = 258 A. Since synthetic polyisobutylene with the same number of chain atoms gives ( P 2 ) ” 2 = 231 A, Boedtker and Doty proposed that the mean configuration of these gelatin chains approaches that of a typical random chain polymer. Further work in this direction was reported by Williams et al. (1954), and Williams (1958) who found an M,,, of 95,000 for the same type of gelatin on the basis of sedimentation equilibrium data. From a combination of
a,,,
STRUCTURE OF COLLAGEN AND GELATIN
97
molecular weight and sedimentation coefficient distributions, a plot of ~ 2 0 versus , ~ M;" was found to be linear, and therefore consistent with random chain behavior. Gouinlock et al. (1955), arrived a t similar conclusions through an analysis of viscosity, sedimentation, and light-scattering measurements of various fractionated, base-processed gelatins. The solution behavior of acid-precursor gelatins deviates quite markedly from the pattern established for their base-processed analogs (Veis and Cohen, 1956, 1957; Veis et al., 1958). At the same molecular weight, acidprecursor gelatins have a lower intrinsic viscosity than do alkali-precursor gelatins. Moreover they exhibit only diffuse changes in light scattering or in electrophoretic mobility through the isoelectric pH region, whereas base-processed gelatins exhibit sharply defined changes over this range. Although the two materials differ in that base-processed gelatin is devoid of amide groups, simple removal of amide nitrogen does not bring the properties into coincidence. Evidently the differences are more fundamental than this. Veis and Cohen believe that acid-precursor gelatins are lateral aggregates of chains held together by occasional cross-links, since this type of compact architecture would be expected to reflect a lower intrinsic viscosity and exhibit less sharply defined configurational changes with pH and ionic strength than the random chain, alkali gelatins. The possibility of chain branching in the molecular structure of gelatin has been investigated by a number of laboratories, following the early suggestions of Mosimann and Signer (1944) and Ames (1952). Courts (1954) measured the a-amino groups of various unfractionated commercial gelatins and estimated i@% to lie between 50,000 and 70,000, in good agrecment with osmotic pressure determinations of Pouradier and Venet (1950). But the later work of Courts and Stainsby (1958) and Courts (1959) indicated that higher molecular weight ox-bone and calfskin gelatins, both alkali- and acid-processed, might well be multichain structures. Thus Mw (light scattering) and [q] (intrinsic viscosity) were virtually independent of the number-average chain weight, M,z, as determined by free a-amino groups. Moreover, Ma for low molecular weight,, acid-processed gelatins was found to be larger than M w , indicating the presence of some chains without terminal a-amino groups. Pouradier (1958) has reported a similar lack of equivalence in M m and the measured carboxyl groups in a baseprocessed calfskin gelatin. The preceding evidence, suggesting that both acid-processed and baseprocessed mammalian gelatins are multichain structures, is entirely consistent with the findings of Grassmann discussed in Section 111 and thus it is pertinent, a t this point, to consider the various types of interchain covalent bonds which might be involved in forming cross-linkages in mature, collagenous tissue.
98
HASRINGTON AND VON HIPPEL
Since collagen is essentially devoid of cystine or cystcine residues, somc type of linkage other than the ubiquitous disulfide bridge must be iiivoked. Bowes and Kenton (1948) found that both the Van Slyke amino nitrogen and the number of titratable amino groups of ox-hide gelatin were lower than expected on the basis of the total lysine content,s and postulatcd the existence of peptide bonds involving t-amino groups. This type of linkage has also been favored by Mechanic and Levy (1959), based upon the isolat,ion of a small amount of the tripeptide N'-(glycyl-a-glutamyl) lysine following mild acid hydrolysis of bovine achilles tendon. Levy et al. (1960) have inferred from their studies on ichthyocol that as much as 40 % of the lysine of this collagen may be involved in N'-lysine bridges, although the recent report of Betheil and Gallop (1960) that 94% of tJhe total lysirie groups in ichthyocol can be guanidinated seems to contradict this conclusion. Ester bonds, imide bonds, and bonds involving side-chain carboxyls of aspartic and glutamic acid have also been proposed as potential branching sites by a number of investigators. Thus, the possibility of interchain type has been considered by Ames imide bonds of the R-CO-NH-COR' (1944). Gustavson (1955a) has postulated the existence of ester linkages in gelatin on the basis of an analysis of the free and amidated carboxyl groups. Evidence of a more direct nature has come from the work of Grassmann et al. (1954) on the citrat,e-soluble collagens of calfskin and Konno and Altmaii (1958) on rat muscle collagen. They find that a small number of covalent, bonds, presumably ester linkages, are reductively cleaved by treatment wit>h lithium borohydride. Gallop et al. (1959) and Bello (1960) have shown that, various gelatins react with hydroxylaminc (pH 9, 40°C) to yield a product containing protein-bound hydroxamate. Gallop and co-workers showed that t,he molecular weight of ichthyocol, calfskin, and acid-processed gelatins was lowered to approximately 20,000 as a consequence of this treatment, without breaking peptide bonds. They have suggested that the hydroxylamine labile bonds are of the ester type and may serve to link together polypeptide segments of similar molecular weight. Some of these hydroxylamine-sensitive ester links may involve the tightly bound carbohydrate moiet'ies discussed in Section I11 (Hormann, 1960). Recently, the presence of y-carboxylglutamyl peptide linkages has also been reported in gelatin. Gallop et al. (1960) esterified a basc-processed commercial gelatin and after cwnvcrsion t20 t>hc protein hydroxamate followed by dinit~rophenylstion,hcat'ed the system in alkali t,o promot,e Lossen rearrangement. of the aspartyl and glutamyl groups. E'rom a n analysis of the products of acid hydrolysis it was inferred that about 31 of the initial glutamic acid residues were involved in y-glutamyl peptide linkages in the esterified protein.
BTRUCTURE OF COLLAGEN AND GELATIX
99
It is probable that differences in maturity of extracted collagens account for a part of the reported variation in the structure of gelatin molecules. The available evidence favors the view that collagen extracted with cold neutral salt solution (tropocollagen) is the most recently formed protein (Highberger et al., 1951; Gross el al., 1955a;Jackson and Fessler, 1955); that collagen extracted with dilute, acidic citrate buffers (procollagen) is a somewhat more advanced morphological form (Orekhovich, 19.52) ; while insoluble collagen represents the most advanced biogenetic stage, which has become sufficiently cross-linked to resist solution. In this view, the degree of cross-linking increaes with the age of the tissue, and the more drastic gelatinizing processes required to solubilize the morphologically advanced collagen structures involve cleavage of cross-linking peptide bonds as well as the splitting of a few bonds along the backbone chain. Orekhovich and Shpikiter (1958a; Orekhovich, 1952) observed that C14labeled glycine was more rapidly incorporated into citrate-soluble collagen than into insoluble collagen, leading them to suggest that the individual procollagen molecules are the precursors of the mature, cross-linked connective tissue lattice. However, in later work Harkness et al. (1954) reported a very rapid incorporation of C'4-glycine into that fraction of rabbitskin collagen which is extracted with mildly alkaline buffer. The rate of incorporation in to citrate-extracted collagen was appreciably lower, and that into insoluble collagen negligible. Jackson (1956, 1957, 1958) observed a similar sequence in his study of the C14-glycine incorporation into the carrageenin granulomata of guinea pigs. The chronological pattern of development of the insoluble, connective tissue lattice from the soluble collagen monomers is also evident from studies of the solubility of various collagen extracts as a function of salt Concentration. It has been clear for a number of years from the work of Gross and his associates (Gross et al., 195Fja; Gross, 1958) that the soluble collagens can be divided into classes on the basis of the salt concentration used in their extraction. More recently, Jackson and Bentley (1960) have demonstrated that the amount of collagen which can be extracted from the induced carageenin granulomata of guinea pigs increases with increaisng NaCl concentration. This finding, coupled with the observation that the most extractable collagen fraction also incorporates C14-glycine most rapidly, led them to propose that a continuous spectrum of molecular aggregates, increasingly strongly cross-linked, exists in the native connective tissue. Although it has been assumed that cross-linking in soluble collagens involves only secondary bonds (Jackson and Bentley 1960), it seems likely that covalent cross-links may well account for the insolubility of the more mature, collagenous tissue. The presence of covalent cross-linkages in the neutral-salt and citrate-
100
HARRING'I'ON AND VON HfPPEL
buffer insoluble collagens is particularly emphasized in the recent studies of Veis et al. (1960), who have examined the sedimentation patterns of such materials after bringing them into solution a t a temperature of 60°C. These solutions exhibit heterodisperse Ultracentrifuge patterns with a t least three major components. The two faster sedimenting components, which have apparent molecular weights of 4 and 12 X 106, are not reduced in weight or amount by hydrogen-bond competing reagents or by acidic or basic environment at temperatures which normally destroy gelatin aggregates. Since the weight of the collagen monomer is thought to be around 350,000, it seems clear that some type of covalent linkages exists between the collagen monomers in the intact fiber structure. Most of the evidence for cross-linking in gelatin comes from work on mammalian material. Fish collagens are in general more readily soluble in dilute salts, suggesting a lower order of covalent bridges between the collagen monomers. This point should be kept in mind when comparing the physicochemical behavior of various gelatins. 0. Gelatins Derived from Soluble Collagens. In Section IV we discussed the denaturation of the asymmetric, rodlike collagen molecules, demonstrating that the viscosity and optical levorotation undergo a precipitous decline at a relatively low and characteristic transition temperature in the presence of certain neutral salts or the hydrogen-bond competing reagents urea and guanidine-HCl. Because of the mild conditions used in such denaturation procedures, it is likely that only secondary linkages are cleaved and we would expect the multichain collagen structure to be dissociated, under controlled conditions, into its component polypeptide chains; providing only that these chains are not held together by covalent, interchain cross-linkages. In Section IV we referred briefly to the sedimentation studies of Orekhovick and Shpikiter (1955a) on the parent gelatin derived from ratskin procollagen, pointing out that a two-component sedimentation pattern is observed when this material is examined in the ultracentrifuge. Qualitatively similar bimodal sedimentation patterns have also been reported for the parent gelatins prepared from the soluble collagens of the swim bladder of the carp (Chun and Doty, 1958), calfskin (Doty and Nishihara, 1958; Pize, et al., 1960) and codskin (Doty and Nishihara, 1958). The relative amounts of the two components appear to vary somewhat, depending on the species, but in general the sedimentation coefficients are quite comparable when measured in identical solvent systems (Table XII). At present only preliminary physicochemical studies have been carried out on the two components. Orekhovich and Shpikiter (1958b) were able to effect a separation of the components from ratskin procollagen by ammonium sulfate fractionation of urea solutions. The isolated fractions each
101
STRUCTURE OF COLLAGEN AND GELATIN
exhibited a single boundary in the ultracentrifuge and diffusion experiments yielded values of D20,w= 3.7 X lo-’ cm2/sec and 2.7 X 10-7 cmz/sec. When coupled with sedimentation coefficients (sZo , w ) molecular weights of 80,000 and 130,000 were calculated for the slower sedimenting (a) component and the faster sedimenting component respectively. The molecular weights of the two components of ichthyocol and calfskin gelatin have been estimated from their respective sedimentation coefficients
(a)
TABLEXI1 Sedimentation Coeficients of the a-and @-Componentso j Vurious Soluble Collagens Collagen source (1) Calfskin
(2) Ratskin (3) Carp swim bladder (4) Dogfish
sharkskin
Solvent
0.15 M Citrate-1.2 M KSCN, pH 3.7 0.15 M Acetate, pH 4.8 Phosphate-1 M KSCN, pH 8 0.15 M Citrate, pH 3.7 0.15 M Citrate, pH 3.7 0.15 M Citrate, pH 3.75
3.85s
5.45s
-1
b
3.24 S
4.41 S
-1
C
4.0 S
5.75 S
-1
d
3.31 S
-
>>I
e
3.15 S
4.3 S
>1
f
3.42 S
5.03 S
-1
g
Ratio of areas under a- and p-peaks [not corrected for Johnston-Ogston effect (194G)1. * Doty and Nishihara (1958). 0 Piez et al. (1960). Ratio obtained from amino acid data. d Orekhovich and Shpikiter (1958a). Determined by comparison with known mixtures. e Gallop (1955b). 1 Chun and Doty (1958). Lewis and Piez (1961). Ratio is corrected for the Johnston-Ogston effect. Q
and the relation between szo,w and aW proposed by Williams, et al. (1954) for a commercial gelatin. Molecular weights of 80,000and 160,000for ichthyocol (Chun and Doty, 1958) and 120,000 and 230,000 for calfskin (Doty and Nishihara, 1958) were obtained in this way. I n the studies of Doty and co-workers, as well as those of Orekhovich and Shpikiter, aqueous KSCN solutions have been employed as solvents in order to minimize association reactions between the gelatin chains. This solvent system gives consistently higher sedimentation coefficients for the a- and 0-components than do simple acetate or citrate buffers (Piez et al., 1960). When the molecular weight,s of
102
HAHHINGTON AND YON HIPPEL
the calfskin components are estimated from the sedimentation coef€icients in citratr buffer using the Williams-Saunders-Cicerelli relation, molecular weights of 80,000 and 160,000 are obtained (Piez et al., 1960). The amounts of the a- and @-componentsobtained are about equal in the parent gelatins derived from the soluble collagens of ratskin, calfskin, and codskin, but in ichthyocol the slower sedimentating a-peak is appreciably larger than the &peak. Moreover, as pointed out in Section IV, the relative distribution of these components can be altered by raising the pH or by prolongrd treatment at a temperature of about 40°C. Under these ronditions, the 0-peak of all of these species disappears and the a-peak iitcreasrs in area, suggesting that the @-component is composed of two polypeptide units similar in size to the a-component, these being held together by a thermal- or alkali-labile bond (Doty and Nishihara, 1958). Differences in the stability of this bond(s) in the different species could account for differences in the a-@distribution. It seems clear that a part of the heterogeneity with respect to size reported for commercially preparrd gelatins must be attributed to both the molecular weight and mass distribution of the a- and @-components.Thus, assuming the weight of the a-component to be 80,000, that of the 0-component to be 160,000, and that dissociation of the collagen molecule yields two a-components and one 0-component as suggested by several authors (Orekhovich and Shpikiter, 1958s; Boedtker and Doty, 1955; Piez et al., 1960), the number average molecular weight, of the mixture is 107,000 and the weight average molecular weight, is 120,000. There seems little doubt that the amino acid compositions of the a- atid @-components differ significantly. Orekhovich and Shpikiter (195%) hydrolyzed the a- and @-fractionsderived from ratskin and demonstratrd, using paper chromatography, that the a-component was richer in hydroxyproline than the 0-component. Similar differences in hydroxyproline content were found by Chun and Doty (1958) for the components of ichthyocol following ethanol-water fractionation. The most complete study of these components to date is that of Piez et al. (1960) who separated the a- and @-components of calfskin parent gelatin on carboxymethyl (CM) cellulose columns. Subsequent amiiio acid analyses of each showed appreciable differences between the two components: the 0-component contained more than twice as much histidine, about half as much tyrosine, and 50% more hydroxylysine, leucine, isoleucine, and valine than the a-component. Proline, hydroxyproline and certain other amino acids also differed by values ranging between 2 and 10 %. The amino acid analysis of the undenatured, purified calfskin collagen was found to be identical with the mean composition of the separated componrnts, indicating, in agreement with the sedimentation patterns, that equal weights of a and 0 are present in the collagen monomer.
an,
aw,
STRUCTURE OF COLLAGEN AND GELATXN
103
Very recent studies on the soluble collagen of ratskin have clarified considerably the relatioilship between the a- and @-compoiients. Orekhovich et al. (1960) and I'iez et al. (1961) have rcported that neutral-salt-soluble collagen gives prcdomiiiaritly the a-component on denaturation, with only a small amount of @-compoiient.Chromatography (I'iez et al., 1961) of this material at 40°C in the preseiice of 6 M urea on CM-cellulose columns reveals the presence of two components, denoted a l and a2, with differing amino acid composition. On the other hand, sedimentation analyses of acid extracted (3% acetic acid) ratskin collagen gave two peaks in the ultracentrifuge with sedimentation coefficients characteristic of the a- and Pcomponents while chromatography of this material (40°C or 6 M urea) revealed four major components. Two of these, a1 and a2, appeared to be the same as the salt-extracted material as judged by sedimentation and chromatographic behavior. Of the remaining two components, one, denoted 01, had a composition equivalent to an equal mixture of a1 and a2, whereas the composition of the other component, 02, was identlical to that of a l . Thus the amino acid analysis, when taken in conjunction with the sedimentation properties of these components, suggests that the a-component] is a mixture of two polypeptide chains of about the same mass but differing composition, while the @-componentconsists of a mixture of two different types of cross-linked chain pairs, i.e., al-a1 and al-a2. Essentially similar conclusions have been reached by Grassmann et al. (1961) on the basis of an analysis of sedimentation patterns of denatured, acid-extracted calfskin collagen. They propose that the formation of the @-componentarises from cross-linking of two a components which may or may not be identical in amino acid composition, while a third component always present in small amount, the y component, represents a structure in which three a-components are cross-linked together. 2. Optical Rotatory Properties
a. Effect of Chain Weight and Composition. At room temperature and above, the optical rotatory characteristics of gelatin are not much influenced by variation in chain weight. Ferry and Eldridge (1949) demonstrated the specific rotation of a series of ossein gelatins in the molecular weight range (aw) 33 t o 72 X lo3 to be virtually invariant (at [a1646 = - 165") above a temperature of 30°C. Similar results have been reported by Saunders and Ward (1958a) for higher molecular weight, fractionated oxhide gelatins above 40°C. A comparison of the rotatory properties of a wide variety of gelatins should consequently be essentially independent of any chain degradation resulting from isolation procedures. The influence of composition can best be evaluated from the careful study of Rurge and Hynes (1959b) who measured the specific rotation of a num-
104
HARRINaTON A N D VON HTPPEL
ber of gelatins a t 40°C following the thermal disruption of the respective collagens. It will be seen from Table XI11 that the specific rotation ([LY];') for these gelatins varies between -109.7' and -135", the increase in levorotatiori paralleling the increase in imino acid content. Assuming a random configuration for the gelatin chain, we would expect the specific rotation t o be obtained by summing over the specific residue rotations of the complete ensemble of amino acids. The residue rotation, [Rlpro,of proline was shown to be close to -250" in Section 11; the residue rotation of glycine, [RIcl, = 0. The residue rotation of L-hydroxyproline is not as well TABLE XI11 Estimation of the Configurational Contribution to Optical Rotation f o r Various Gelatins at 40°C Gelatin source
HY- GlyProlinea droxy- cinea proline
References
___
Calfskin Rntskin Perch swim bladder Dogfish sharkskin Carp swim bladder Cod swim bladder Codskin
138 130 118 99.4 116 103 99.4
94 93 81 57.G 81 57 53
320 351 333 340 325 333 345
- 140" - 135" -127.5"
-27" -27" -21" -19"
- 120" - 124"
- 16"
-116" -110"
-10"
-14"
Residues of amino acid per 1000 total residues. Assuming mean residue weight is 91. c Piez and Gross (1960). d Doty and Nishihara (1958). 0 Harrington (1958). I Burge and Hynes (1959a). Burge and Hynes (1959b). h Lewis and Piez (1961). i von Hippel (unpiiblished). a 6
established as that of L-proline. However, the specific rotation of poly-Lhydroxyproline in concentrated aqueous LiBr is - 168" and, reasoning by analogy with the studies on poly-L-proline I1 in this solvent, we would expect the structural contribution of the chain to be eliminated. Correcting this specific rotation for the residue weight of hydroxyproline gives [R]Hypro= - 190". Heynes and Legler (1960) have recently estimated the residue rotation of hydroxyproline from the specific rotation of the tripeptide carbobensoxy-Ala.Gly.Hypro-NHz at -222". In the calculation to follow we will assume R,,,,, = -200". I n view of the absence or trivial presence of other amino acids with unusual residue rotations, the mean residue rotation of all other amino acids in gelatin may be taken as - 110"
STRUCTURE OF COLLAGEN AND GELATIN
105
(Schellman and Schellman, 1958) and we may proceed to an estimation of the specific rotation based on the composition. 'The specific rotation of a long polypeptidc chain which is locked into an asymmetric configuration may be expressed as :
where
[Ri]is the intrinsic residue rotation of the ith amino acid residue;
MRW, the mean residue weight; n, the number of residues in the chain; and [a] o,configuration , the
structural contribution of the chain. Calculation of the expected specific rotations from Eq. (9) and the known amino acid compositions of the gelatins in Table XIII, demonstrate that a significant amount of left-handed configuration remains in the polypeptide chain of each species, even at temperatures of 40°C. Harrington (1958) attributed this residual optical asymmetry to the existence of elements of a poly-L-proline II-type configuration imposed by the presence of prolineproline sequences. When two imino acid residues occur contigfuously in a polypeptide chain, the orientation of nine backbone bonds along the chain is fixed in the left-handed poly-L-proline II-t,ype configuration assuming restricted rotation about, the
c.-c
//
0
Bond (ii)
(ii) bonds (see von Hippel and Harrington, 1959). These poly-L-proline I1 "nuclei" may be of special significance in the mutarotation of gelatin at low temperature (see below). b. E$ect of Salts. It has been known for many years that a number of neutral salts have a rather profound effect on the optical rotation of gelatin (Stiasny et al., 1925; Katz and Wienhoven, 1933; Carpenter, 1938). In their studies, Carpenter and Lovelace (Carpenter, 1927; Carpenter and Kucera, 1931; Carpenter and Lovelace, 1935a, b, 1936,1937) examined the effect of a wide spectrum of electrolytes at concentrations up to 4 M , and observed that the specific rotation, [a]: of calfskin gelatin decreased from - 134.5' to values ranging between - 90" and - loo", the magnitude of the change depending on the nature of the salt. The capacity of ions t o change the specific rotation followed a Hofmeister series, with lithium cations and thiocyanate anions exhibitng the most striking effects. Under these conditions of varying electrolyte environment, the optical rotatory dispersion of gelatin follows a one-term Drude equation over the visible region of the spectrum, with an unchanging Drude dispersion parameter, A, = 220 mp. At high concentrations of aqueous lithium bromide (8-12 M ) , may
10G
HARRINGTON AND VON HIPPEL
decrease to values as low as -35”, and careful dispersion measurement’s reveal an accompanying decrease in A, ranging between 2 and 22 m p , depending on tbe gelat,in species (Harrington, 1958). Alt,hough thc dath are not extensivc, the magnitude of these changes appears to parallel the imino acid content of the gelatins measured, being greatest for calfskin and least for ichthyocol gelatin. Neutral salts have been shown to have striking effects on other polypeptide chains as well. The rotatory parameters of oxidized ribonucleasr and clupein, which are known to exist as unfolded chains in aqueous solution, change to those characteristic of a-helical proteins in high conceiitrations of LiBr (Harrington and Schellman, 1957). These observations led tJo the suggestion that LiRr, and other similar salts with unusually high activity coefficients, attenuate the disruptive effect of water on hydrogen-bonded peptide structures by lowering the water activity. The findings of Harrington and Schellman have recently been reinforced by the deuterium-exchange studies of Stracher (1960), who has shown tjhatj oxidized ribonuclease assumes a partially-folded, hydrogen-bonded configuration i; this medium. Stracher also found, however, that comparable concentrations of NaBr seem to have approximately the same effect as LiBr on the deuterium-exchange properties, even though NaBr exerts a relatively small effect on the activity of water. A similar correspondence between thesc two salts was also observed in the optical rot,atory experiments of Rigclow and Geschwind (1961). It seems probable that these salts produce a profound configurational change in the polypeptide chain, but the fundamental mechanism involved may be somewhat more complicated than originally appreciated by Harrington and Schellman. There can be little doubt that a marked structural change in polyL-proline I1 occurs in the presence of high concentrations of LiBr, and NaSCN, since the intrinsic viscosity of this substance is strikingly depressed in the presence of these salts. This cannot be a simple “solvent effect,” since the effect of aqueous lithium bromide on the optical rotation of simple derivatives of proline is relatively small, amounting to a change of a t most 5 % in the absolute value of the specific rotation (Steinberg et al., 196Ott). Similar minor changes in optical rotation were reported by Bigelow and Geschwind (1961) for several other low molecular weight substances in the presence of aqueous LiBr solutions. To the present authors, the most likely explanation of the optical rotatory changes in gelatin, poly-L-proline 11, ribonuclease, and clupein is that these reflect deep-seated changes in chain configuration brought about through fundamentally similar mechanisms. This is believed to arise from a modification in the solvent-chain interaction which is mediated by the salts. In the case of poly-L-proline 11, we have already given evidence t.hat LiBr and NaSCN “unlock” the bond (ii) linkages, allowing free rotation about, t,hcse bonds. This process is thought
STRUCTURE OF COLLAGEN AND GELATIN
107
to arise from a modification of the solvation of the carbon yl-oxygen atom, the solvent binding in the peptide region in the absence of salt and thus preventing rotation of the pyrrolidine rings about the backbone. Clearly LiBr and NaSCN may be modifying the peptide group solvation through quite different mechanisms, since it is well known that the lithium and thiocyanate ions have markedly different effects on the activity of water. Similarly, the optical rotatory changes in gelatin in the presence of these same salts may well rest on the attenuation or modification of the solvent binding. In gelatin, we might expect the bond (ii) linkage of the prolinehydroxyproline sequence to be unlocked, thus eliminating any residual poly-L-proline II-like structure. The distribution of proline residues along the gelatin peptide chain would prevent the formation of an extensive ahelical configuration, analogous to that observed in the oxidized ribonuclease and clupein in the presence of LiBr. This is also consistent with the rotatory parameters. Although the specific levorotation of gelatin is greatly reduced in concentrated LiBr, A, exhibits only a slight decrease, similar t o that observed in poly-L-proline I1 in this solvent, but opposite in sense to that observed for clupein and ribonuclease. Indeed, the very low specific levorotation of gelatin in concentrated aqueous LiBr (about 60" below the specific rotation expected for a random chain, see Table XIII) suggests that the configuration is right-handed, in contradistinction to the left-handed sense of the chain as it exists in the collagen structure. It is also apparent from this discussion that the stability of the threestranded collagen molecule in aqueous solution would be drastically lowered in the presence of a high concentration of these neutral salts, the integrity of the individual, left-handed poly-L-proline II-type helices of the macromolecule being distorted and destroyed as the bond (ii) linkages between contiguous pyrrolidine rings are unlocked. These regions, in addition to bcing the seat of the left-handed structure, must confer considerable stability on the helix.
3. Nonaqueous Solvent Systems The bchavior of gelatin in mixed organic solvent systems h i s bccii rcported by Veis and Anesey (1959) and Steinberg et al. (1960a). Veis aiid Anesey have observed that the addition of dimethylformamide (DMF) to a formic acid solution of pigskin gelatin lowered the specific rotation from [alD= - 116" to [aID= -58" (in 5 % formic acid-95 % dimethylformamide). Similarly, Steinberg et al. observed an immediate decrease in [aIDon dilution of a formic acid (FA) solution of bovine gelatin with n-propanol (8:l propanol-formic acid, v/v). No further changes in rotation were observed on standing. From rotatory dispersion data, A, was found to decrease from 217 to 203 mp in the mixed solvent system. The intrinsic viscosity of gelatin in formic acid decreases on addition of
108
IIARRINGTON AND VON HIPPEL
dimethylformamide, reaching a minimum value a t a ratio FA/DMF = 1. Further addition of dimethylformamide leads to an increase in intrinsic viscosity (Veis and Anesey) . Since light-scattering studies indicate that the molecular weight of gelatin remains unchanged throughout these transformations, it appears that both the rotation and the viscosity changes reflect alterations in the configuration of individual gelatin molecules. The solvent system employed by Veis and Anesey has been shown to lead to random coil -+ a-helix transformations in synthetic polypeptides (Yang and Doty, 1957) but the decrease in A, and the anomalous minimum in the viscosity versus solvent plot suggest that in the case of gelatin another type of structure may be forming. Veis and Aniiescy propose a right-handed helix of the poly-L-prolinc I-type, generated through trans 3 cis-isomerizations a t the proline-proline peptide bonds. Steiriberg et al. also believe that the rotatory changes result from induced rotations of neighboring pyrrolidine rings about the backbone, but suggest that this occurs at bonds (ii). It may well be that increased rotational freedom a t both linkages is involved in generating the new structure.
B. The Molecular Properties of Gelatin at Low Temperature When gelatin solutions are cooled to low temperature, striking transformations are seen in a number of physical properties. The viscosity increases with time, and if the protein concentration is above a critical level the system soon sets to an elastic gel. Dilute isoionic solutions do not exhibit the gelling phenomenon, but the viscosity increases continuously over a prolonged period of time at low temperatue and light-scattering studies demonstrate an association between gelatin chains, leading to molecular weights of the order of 40 X loe (Boedtker and Doty, 1954). I n the presence of salt or at pH values removed from the isoionic point, lower molecular weight aggregates are formed. These transitions are accompanied by substantial changes in optical rotation, the specific rotation increasing from [a],= - 120" to values approaching those characteristic of native collagen [a],= -350" to -400"; (Smith, 1919; Ferry, 1948a; Ferry and Eldridge, 1949; Robinson, 1953; Cohen, 1955; von Hippel and Harrington, 1959; Flory and Weaver, 1960). Careful measurements during the gelling process have demonstrated that the volume also decreases slowly with time (Derksen, 1935; Heymann, 1936; Neiman, 1952, 1954; Flory and Garrett, 1958). The over-all sol-gel transformation is reversible; the rigidity, levorotation, volume, and heat content changing in unison through a fairly well-defined temperature range (Smith, 1919; Pleass, 1930; K a t a , 1933; Holleman, et al., 1934; Derksen, 1935; Ferry, 1948b; Ferry and Eldridge, 1949). 1. X-ray Di$raction and Infrared Studies Cold gelatin gels formed by evaporation exhibit a rather high degree of order, with X-ray reflections at 2.8, 7.8, and 11.5 A, and in addition, an
STRUCTURE OF COLLAGEN AND GELATIN
109
amorphous halo a t 4.4A (Hermann et al., 1930; Katz et al., 1931; Katz and Derksen, 1932). It will be remembered that strong 2.8 and 1 1.5 A reflections are also observed in native collagen. These reflections disappear from the gelatin gel on heating above the transition temperature, but slowly reappear on cooling. Gels which havc been oriented by stretching display X-ray diffraction patterns strongly resembling those of collagen, with characteristic meridional reflections at 2.86 A and equatorial spacings of 10 to 16 A (Hermann et al., 1930; Derksen, 1935). When concentrated gelatin gels are stretched, followed by heating, they show a well-defined shrinkage temperature with characteristics quite comparable to the thermal shrinkage of collagen (Hirai, 1953). Cold evaporated gelatin gels which have been treated with neutral salts also exhibit a sequence of X-ray diffraction patterns paralleling those obtained for native collagen under similar environmental conditions (Ramachandran, 1958). Noteworthy structural similarities between cold gelatin and native collagen may also be inferred from infrared studies. Robinson and Bott (19,51) found the N-H stretching frequency of a film prepared by evaporation of a hot (40°C) gelatin solution to be 3310 cm-I, whereas in a film evaporated at room temperature this band was shifted to 3330 cm-I, the value characteristic of collagen. Comparable results have been reported by Bradbury et al. (1958) who observed the infrared spectrum of cold-cast gelatin t o be intermediate in character between native and completely denatured collagen. Furthermore, when cold-cast gelatin films are stretched, the resulting infrared dichroic ratios at 3330 cm-I (N-H stretch) and 1650 cm-1 (C=O stretch) have the same sense as those characteristic of native collagen, but no dichroism is detected in hot evaporated gelatin films; again indicating the lack of structural order in the polypeptide chains under these conditions. Taken in conjunction, the physical data summarized above strongly suggest that the crystalline structure of collagen is partially regenerated on cooling gelatin. This view is supported by the recent electron-optical studies of Veis and Cohen (1960) and Rice (1960), who have demonstrated that many of the morphological features of native collagen return on cooling gelatin under controlled conditions.
2. Optical Rotatory Properties The striking change in levorotation seen on chilling warm gelatin solutions must be judged one of the most interesting properties of this system. At concentrations of the order of 0.5 % gelatin and above, the mutarotation is generally accompanied by gelation and the apparent relationship between mutarotation and the gelling phenomenon has consequently been investigated by a large number of workers (see Ferry, 1948a). Certainly one of the most careful early studies in this area was that of Smith (1919) who
110
HARRINGTON AND VON HfPPEL
found that a t temperatures of 35°C and above and a t gelatin concentrations between 1 and 7%, the specific rotation of aqueous solutions of ossein gelatin = -120") was virtually independent of temperature and protein concentration. At low temperatures (< 15"C), the specific levorotation was seen to change over a prolonged period of time a t a rate inversely proportional to the concentration of gelatin, approaching a nearly constant specific rotation of [aID= -313". On raising the temperature stepwise between 15" and 35"C, the specific rotation leveled off a t progressively lower exhibiting a slight concentration-dependence at any values, with [all, temperature. Smith concluded from these studies that two forms of gelatin exist; one stable above 35°C (sol form) and one stable below 15°C (gel form), and that at temperatures intermediate between 35" and 15°C these are in equilibrium. Similar differences between the properties of gelatin at high and low temperature were revealed by an investigation of dried gelatin gels. Gelatin dried at temperatures above 35°C showed a n [aID = -120", whereas samples dried at temperatures below 15°C displayed a much higher levorotation, approximating [a] = -750". The early proposals of Smith have been supported and extended by the work of Robinson (Robinson and Bott, 1951; Robinson, 1953) who found [aIDof a cold evaporated gelatin film to be about -1000", whereas a hot, evaporated film showed [aI0 = -128". From these experiments, and his infrared absorption and dichroism studies on gelatin films, Robinson has suggested that gelatin exists as single molecules in a configuration which cannot form interchain hydrogen bonds a t temperatures greater than 35°C. At lower temperatures, Robinson assumed that a unique configuration (the collagen-fold configuration) develops which can form interchain hydrogen bonds. On the basis of her extensive studies on the optical rotation and optical rotatory dispersion properties of ichthyocol gelatin, Cohen (1955) reached similar conclusions, attributing the increase in levorotation ([a]~6 =0 -110'; = -350") observed on cooling gelatin solutions specifically to the development of a helical configuration along the polypeptide chain. A perusal of the data of Smith discloses that the specific rotation of 24hr, low temperature gelatin is remarkably insensitive to protein concentration, in contradistinction to the direct relationship between mutarotation and chain association which some workers have assumed in the past (Kraemer and Fanselow, 1925; Katz, 1933). The independence of the two processes is also indicated by the investigations of Ferry and Eldridge (1949). On chilling ossein, alkali-processed gelatin solutions of various concentrations (2-6 % protein), each solution attained virtually the same specific rotation ( [ 0 1 ] 6 6 ~ ~= -375") after 24 hr. Moreover, the melting-out profiles of all of the solutions were essentially coincident, with [a]:46 declining
[a]r
STRUCTURE OF COLLAGEN A N D GELATIN
111
from -265" to - 120" over the temperature interval 15" to 30°C. In these studies, the terminal specific rotation of degraded, low molecular weight gelatins maintained at low temperature varied with the number-average molecular weight; but the specific rotation of various mixtures of two different gelatin fractions = 17 X lo3 and = 44 X lo3)was found to be additive in weight Concentration. Ferry and Eldridge concluded that the change in optical rotation accompanying gelation was due primarily to an intramolecular process within individual gelatin molecules and suggestcd the formation of intramolecular cross-linkages or, alternatively, some type of chain rearrangement. The invariance of the ultimate value of specific rotation of chilled gelati11 with concentration has also been demonstrated by the work of Cohen (1955) on ichthyocol gelatin and of Flory and Weaver (1960) on rat-tail tendon gelatin. Von Hippel and Harrington (1960); Harrington and von Hippel (1961) have followed rotatory changes in chilled ichthyocol gelatin at very short wavelengths (A = 265-313mp) and thus have been able to measure terminal specific rotations at gelatin concentrations nearly two orders of magnitude below those investigated by Smith, and Ferry and Eldridge. Again [a],was shown to be nearly independent of concentration, although the final reduced viscosity varied about sixfold over a comparable concentration range. In substance, then, the mutarotation phenomenon reflects the development of a specific type of structure along each gelatin chain, the formation of which is independent of chain association, at least in the initial stages. In view of the close correspondence between many of the physical properties of cold gelatin and collagen, we assume that the ordered structure regencrated along each chain is that of a poly-L-proline II-type helix. Although the mutarotation of dilute gelatin at low temperature (3°C) is apparently complete in 24 hr, careful measurement reveals that the specific levorotation continues to increase very slowly over a period of many days-paralleling the gradual increase in Viscosity observed during this interval. In fact mutarotation is apparently not complete even after 28 days (Harrington and von Hippel, 1961) and it seems clear that a specific association between gelatin chains may lead to an increased ordering of the individual poly-L-proline I1 helices and consequently an incremental increase in levorotation. It will be remembered that destruction of the highly crystalline collagen structure leads to a change in [alD of about f284". The change in [ a ] ,on cooling dilute gelatin amounts to only about 5 0 4 6 % of this value (Flory and Weaver, 1960; Harrington and von Hippel, 1961) consistent with that expected for a partially disordered strueture. The additional development of the poly- proli line II-type structure on association of chains can be inferred from Fig. 23. After 24 hr at 3°C the
(an
a,,
112
HAHRINGTON AND VON HIPPEL
specific lcvorotation of ichthyocol gelatin (1.67 mg/ml) has recovered about 64% of the ordered collagen structure as measured by optical rotation. On standing 28 days a t this temperature the recovery is about 80%.
TEMP. (OC.)
FIG.23. Specific rotation of ichthyocol collagen and gelatin at 313 mp a s a fuiiction of temperature. Collagen concentration = 1.14 mg/ml, gelatin concentration = 1.67 mg/ml. v, gelatin, after 24 hr at 3°C; A, gelatin after 6 days at 3°C; 0, gelatin after 28 days a t 3°C; 0, native soluble collagen. (From Harrington and von Hippel, 1961.)
Another noteworthy feature of Fig. 23 is the greater breadth of the mehingout profile of chilled gelatin compared t o that of collagen, indicating a lower degree of order and perhaps near neighbor cooperation in the gelatin structure (Schellman, 1955, 1958; Zimm and Bragg, 1958, 1959; Gibbs arid DiMarzio, 1958). Yet the threshold temperature, T , ,is identical for the two systems, revealing the fundamental similarity of their basic molecular
STRUCTURE OF COLLAGEN AND GELATIN
113
architecture. The significance of this relationship has been particularly emphasized by Flory and Garrett (1958; Garrett and Flory, 1956). 3 . Kinetics of Mutarotation
When concentrated gelatin solutions (> 1 %) are held a t low temperature, the levorotation increases rapidly over a period of several hours, after which the rate declines sharply to a comparatively low value. Smith (1919) found the [a],versus time profile to correspond closely to the behavior expected for a second-order reaction , the over-all rate varying inversely wit,h protein concentration. Qualitatively similar kinetic patterns are observed in gel rigidity (Ferry, 194813). During the initial stages of gelation the rigidity increases very rapidly, this phase leading into a prolonged interval in which the rigidity changes a t a greatly reduced rate. Although Smith interpreted his mutarotation kinetics in terms of a second-order reaction, the over-all time-dependent change, both in optical rotation and rigidity, could rcsult from combination of a fast process followed or accompanied by a slow process. That this is indeed the case may be seen from the results of two recent, studies on the mutarotation of gelatin at low protein concentrations. Flory and Weaver (1960) found the rate of mutarotation of rat tail tendon gelatin to be independent of concentration (in the range 0.5 to 4 mg/ml) at temperatures between 5°C and 23°C. Von Hippel and Harrington (1960) reported a similar invariance in rate of mutarotation of ichthyocol gelatin over the range 0.1 to 1.7 mg/ml (at 3.9”C). It was demonstrated in the latter study that the over-all process consists of two phases (Harrington and von Hippel, 1961) of which the primary phase is independent of protein concentration and is completed in about 15 hr at 3.9”C1while the secondary phase is, as we have seen, a concentration-dependent association between gelatin chains and leads, in this concentration range, to an incremental increase in rotation which takes a very long time to go to completion. At high concentrations the chain association reaction would be expected to be markedly increased in rate, leading to a compression of the [a],versus time curve which would mimic a second-order process. Flory and Weaver (1960) have advanced a mechanism for the reversion kinetics, in which they postulate the formation of an intermediate (involving the intramolecular rearrangement of a single gelatin molecule) as the rate-determining step. The intermediate, which may be a helix of the poly-L-proline 11-type, is converted rapidly to a three-chain compound helix a t a rate “sufficiently rapid to have no effect on the over-all rate.” The scheme may be represented as follows: ki’
C
I ‘ b
L
h’
h
%(H)
114
HARRINGTON AND VON HIPPEL
where C, I, and H represent t,he random coil, single-chain intermediate, and nat,ive, three-chain collagen helix, respectively. The concentration of intermediat,e is assumed to be very small compared to the concentration of C, so that t,he over-all process should be first-order (with rate, R' = Icl' [C]). The second step of the process consists of the lateral aggregation of the intermediate species and is thought t o he both comparatively rapid and easily reversible. A crucial question with respect to the Flory-Weaver scheme is t,he rat,e of the second step, i.e., the lateral association between chains t,o form t,he native three-stranded compound helix. This process should he strongly concentration-dependent, a change in concentration of tenfold leading tjo a change of a hundred- to a thousandfold in rate. In fact we have seen that, at, concentrations between 1 and 7 % (Smith, 1918), t,he over-all rate of mutarotation is strongly influenced by concentration. On the other hand, the lack of a concent,rat,ion-dependenceat, the very low prot,eiri concent,rations used by Flory and Weavcr (1960) and Harringtm and von Hippel (1961) suggests that this st3ep,t'he lateral associat>ionhet,weeii chains, has been reduced in rate to a comparatively low value. Further evidence in support of this supposition comes from the viscosity studies at, low concentration. At concentrations of less than 1.5 mg/ml, the primary st,ep of mutarotation is completed in 24 hr at 4°C. During this time interval the reduced viscosity, qsPic has increased from -0.30 to -0.65 dl/gm. In cont,rast to this latter value, the reduced viscosiy of the three-chain compound helix is about 14 dl/gm (Table VII). Clearly the molecular structure present a t the end of the primary phase cannot be the three-stranded compound helix of collagen. These considerations lead us to an alternative mechanism, which had been proposed earlier by the authors of this review (von Hippel and Harrington, 1959, 1960; Harrington and von Hippel, 1961). We assume t,hat the first-order kinetics of the primary phase reflects the development, of a configuration of the poly-L-proline II-type along each chain, and that this is a stable configurat,ion at low temperature. The chain may exist either as a single helix or as helical segments separated by random chain elements. Helical segment,s of neighboring chains are able to pack laterally, giving (locally) the highly ordered interchain hydrogen-bonded structure found in native collagen. Lateral association between chains allows a further ordering of the noncrystalline regions of each chain. The difficulty with this proposal is the requirement for intramolecular stabilization of the helical elements. As we have seen, the left-handed helix of poly-L-proline I1 is stabilized in aqueous solut,ion a s a result of the restraints imposed a t the peptide linkage and the
115
STRUCTURE O F COLLAGEN AND GELATIN
0 Bond (ii)
bonds. I n the gelatin chain, however, free rotation is theoretically possible about every third backbone linkage in the non-imino acid residues. It is highly unlikely that a systematic set of peptide hydrogen bonds, acting in a
- 1900 -1700
-1500
n
;--I300
23
-1100
- 900 -
- 700
0
10
20
x)
TEMPERATURE
PC)
40
50
FIG.24. The specific rotation of ichthyocol gelatin a t 313 mp as a function of temperature. Protein concentration = 1.67 mg/ml. Solvent: 0 , 0.5 M CaClz in DzO; 0 , 0.5 M CaClz in HzO. Samples on solid curves were held a t 3°C for 24 h r after quenching from 45"C, samples on dotted lines were held for G days after quenching. (From von Hippel and Harrington, 1960.)
inaiiiicr analogous to that of tJhea-hclix, could stabilize this stmcture. This hydrogen-bonded arrangement is ruled out both by the lack of peptide hydrogen donors in the imino acid residues and by the steep pitch of the poly-L-proline 11-type helix. Nevertheless, some type of hydrogen bonding is indicated, since no observable time-dependent changes in optical rotation are observed on cooling aqueous gelatin solutions in the presence of high concentratioiis of the hydrogen-bond-competing reagents urea or guanidineHC1. Moreover, when the melting-out profiles of cold (24 hr a t 3°C) dilute ichthyocol gelatin in DzO are comparcd to those in HZO (Fig. 24), the mid-
116
IIARRINGTON AND VON HIPPEL
point of the transition is found to be elevated about 3.7”C (Harrington and von Hippel, 1961). A similar temperature differential was found by Hermans and Scheraga (1959) in comparing the “melting” temperature of the hydrogen-bonded secondary-tertiary structure of ribonuclease in these two solvents. The explanation for the elevation in temperature advanced by these authors is that the -0. . . H peptide hydrogen bond in HzO is less stable than the corresponding 0 . ..D peptide hydrogen bond in DzO. This suggestion is supported by X-ray studies on simple crystals, which have demonstrated an appreciable shift in hydrogen bond length on replacement of hydrogen by deuterium (Gallaghcr, 1959). The change in bond length, which is thought to be primarily due to the difference in zero point vibration energies of the two isotopes, is rclatcd to the type of hydrogenbond and may occur either as u coritractiori or elongation of the bond, depending on the original H-bond length. On the basis of these arguments, we are led to the possibility that u systematic set of hydrogen bonds involving water molecules may develop along the geltttin chain at low temperature. We have in mind specifically the formation of doubly hydrogen-bonded water bridges between adjacent carbonyl oxygen atoms, these forming a cooperative set along the chain and serving to stabilize the poly-L-proline II-type configuration. Evidence for the involvement of water as an integral part of the structure of collagen has been presented in Section IV. We may recall here that the work of Burge et al. (1958), Esipova et al. (1958), and Bradbury et al. (1958), has provided evidence that water molecules will fit into the geometry of the chains of collagen, forming hydrogen-bonded bridges between adjacent carbonyl groups without severe strain. We should also note the extraordinarily strong affinity for water exhibited by imino residues, as shown by the infrared studies of Blout and Fasman on poly-L-proline (1958). Although the rate of mutarotation of cold dilute gelatin is independent of protein concentration, kinetic analysis reveals an exponential dependence of d[a]/dt on the concentration of chain elements in the unfolded form. derived from Thus van’t Hoff plots of log (d [ a ] / d t )versus log ([& -[aI1), the general equation:
are linear over about 80 % of the intramolecular phase of the reaction, with n = 2.2 (f0.15) independent of protein concentration or temperature (see Table XIV). The apparent negative temperature dependence of mutarotation is another striking aspect of the kinetics which is of signal importance in
117
STRUCTURE OF COLLAGEN AND GELATIN
understanding the mechanism of the gelatin -+ collagen-fold transition (Smith, 1919; Flory and Weaver, 1960; von Hippel and Harrington, 1960). Flory and Weaver found the rate of mutarotation of rat-tail tendon gelatin to increase over a hundred-fold when the temperature was decreased from 23°C to 5°C. It was also observed that a logarithmic plot of the half-time of mutarotation against the reciprocal of absolute temperature (log [tx] versus 1/T) was nonlinear over this temperature range, the magnitude of the (negative) activation energy' of mutarotation increasing with temperature. A large apparent negative activation energy (- - 30 kcal/mole) TABLE XIV Kinetics of Optical Rotatory Changes During the Gelatin -+ Collagen-Fold Transition Gelatin
I. Ichthyocol
11. Calfskin
SolventD
H2 0 Ha 0 H2 0 H2 0 H2 0 H 20 H20 H20 DzO D2 0 D2 0 D2 0 H2 0 D2 0
Over-all average:
Concentration Tern erature Order of Reaction6 (mg/ml) PC, (4 1.33 1.33 1.33 1.33 1.28 0.64 0.32 0.16 1.57 1.57 1.57 1.57 1 .?O 2.0
3.70 5.20 8.00 11.40 8.00 8.00 8.00 8.00 3.70 5.05 8.00 11.35 3.7 3.7
1.9 2.1 2.4 2.3 2.3 2.0 2.1 2.4 2.2 2.1 2.2 2.2 2.2 1.9 2 . 2 f 0.15
All solutions 0.5 M in CaClz . van't Hoff plot.
* Slope of
over the temperature interval 3°C to 12"C, has also been reported for ichthyocol gelatin (Harrington and von Hippel, 1961). The magnitude of the negative activation energy and its variation with temperature suggest a phase transition, similar to a crystallization process developing from preformed or rapidly formed nuclei (Flory and Weaver, 1960; Beckrr and Doring, 1935; Flory, 1949; Flory and McIntyre, 1955; Price, 1959; LauritZen and Hoffman, 1959). Flory and Weaver have shown how their mechanism (see above) could lead to a negative temperature coefficient for the reversion kinetics. Since the intermediate, I, consists of an ordered configuration, its entropy should be appreciably lower than that of C, the random coil. The over-all (C -+ H)
118
HARRINGTON AND VON HIPPEL
enthalpy change is negative (approximately 1.4 kcal per peptide unit), and Flory and Weaver assume that part of the enthalpy should be lost in the first step of the process, the formation of I. This result, coupled with the assumption of an unstable intermediate of ordered configuration, leads t o a negative heat of activation and a large positive free energy of activation. Crystal growth is assumed to arise from nuclei consisting of helical segments constructed t,hrough the joining of three primary helix intermediates, I, of a critical length of n residues. The apparent intramolecular nature of mutarotation has prompted the present authors tjo examine the kinetics of t’he process in terms of a one-dimensional crystallization along individual gelatin chains (Harrington and von Hippel, 1961). According to this view, the crystal nuclei would be small segments of the poly-L-proline II-type helix which are residual above the transition temperahre, T,,, , or, alternatively, which form very rapidly on lowering the temperature below T, . If it is assumed that crystal growth is propagated a t constant velocity from these nuclei, the apparent order of the kinetics (n = 2.2 f 0.15) can be easily derived. The analysis suggests that the nuclei are grouped in clusters along the gelatin chain. The overriding experimental consideration which led both Flory and Weaver (1960) and the present authors (von Hippel and Harrington, 1959, 1960; Harrington and von Hippel, 1961) to postulate a single-chain intermediate in their mechanisms for collagen reformatlion from cold gelat,in, was the observation that the mutarotation process appeared to be independent of concentration, in marked contrast to properties attributed to interchain interactions as measured by viscosity and light scattering. However, the evidence already discussed in Section I11 and the present section strongly suggests the presence of interchain covalent cross-linkages, a t least in some collagens and gelatins. This raises the possibility that interactjions between cross-linked chains might, nevertheless, be involved in the generation of the collagen-fold. Such a process would still, of course, be intramolecular and so could not be ruled out on the basis of the observed concentration independence of tJhe primary step. These arguments suggested that an investigation of “de-esterified” gelatins might be helpful. As pointed out in Section 111, Gallop et aE. (1959) showed that &w of both ichthyocol and calfskin gelatins is reduced to -20,000 on treat,ment with aqueous hydroxylamine (pH 9, 40°C). Such treatment breaks estertype linkages, but has been shown to have no effect on peptide bonds. Thus for studies of the mutarotation process de-esterified gelatin would have two advantages : (1) the probability that cross-linked multichain units are involved in the process should decrease with decreasing moelcular weight, and (2) the bonds which are broken by the hydroxylamine treatment might well be the postulated cross-linkages.
119
STRUCTURE OF COLLAGEN AND GELATIN
A recent examination of the mutarotation phenomenon in de-esterified gelatin (von Hippel and Wong, 1961) has shown that this process is essentially unchanged from that observed using untreated gelatins, exhibiting similar large negative temperature coefficients and apparent energies of activation, the same order of the reaction from van't Hoff plots (n = 2.0 f0.2; compare with Table XIV), and a similar response to alterations in the ionic environment. Moreover, tjhe number-average molecular weight (estimated from protein-bound hydroxamate) and the weight-average molecular weight (by short-column equilibrium ultracentrifugation) are closely similar, as expected for material consisting primarily of single polypeptide chains of roughly comparable length. TABLE XV Rate of Formation of the Collagen-Fold i n Different Gelatins, Following Quenching to 3.7%' Gelatin
Dogfish sharkskin Ichthyocol Calfskin a
Total imino acid content Transition (residues/ :ERp::tj 1000) 158a 197* 232b
11 18 23"
Initial rate dedmin) In H ~ O
In D ~ O
0.39 4.8 13.1
0.92 13.2 27.7
[d:l8
(final) In Hzo 1940 1900 1972
Piez, personal communication. and Gross, 1980. Smith, 1919.
* Piez
Another revealing feature of the mutarotation process is the cf!fect of the solvent environment on the rate. Harrington and von Hippel (1961) have shown that the initial rate of mutarotation of gelatin in DzO is markedly accelerated over that in HzO. At 3.7"C, for example, the initial rate of mutarotation of ichthyocol gelatin in DzO is over 2.5 times that in HzO. Similltr ratios have been observed with dogfish shark and calfskin gelatin (see Table XV). These findings may be explained on the basis of the large, negative temperature cxfficient of the mutarotation process and the finding that the melting temperature in DzO is elevated by approximately 3.7"C above that in HzO (see Fig. 24). Since measurement of the rate of helix formation a t a fixed low temperature involves a larger temperature differential between the experimental temperature and T, in the DzO solvent, the rate in DzO would be expected to be greater than that in HzO. In fact, if the difference in T , in the two solvent systems and the apparent energy of activation of the process are taken into account, the rate in DzO becomes essentially identical to that in HzO.
120
HARRINGTON AND VON HIPPEL
Other methods of tampering with the solvent environment lead to comparable results. In the study discussed above, all the experiments were carried out in an identical ionic environment, namely neutral 0.5 M CaC12. Recently von Hippel and Wong (1961) have examined the effect of varying the ionic milieu of the gelatin chains, and find that the initial rate of mutarotation is a very sensitive function of both ion concentration and ionic composition. For example, the initial rate is approximately tenfold larger in 0.025 M CaClz than it is in 0.5 A4 CaClz (see Fig. 25), and the melting profile for both ichthyocol and calfskin collagen in 0.025 M CaClz is found to be Temp. = 4. C Cone. = 1.6 m*lm~ 0 CoClt (PH 7 ) A C a C I e 4 Glyclne (PH 25) V C o C l o + Olycin. (pH 10.5)
ionlo
Strcngth
FIG.25. Initial rate of rnuLarotation of ichthyocol gelatin cooled to 4°C at zcro time, as a function of ionic strength (CaClZ). (From von Hippel and Wong, 1961.)
shifted upward several degrees when compared to the profiles obtained in 0.5 M CaCl2. The effect of a variety of cations and anions on the rate of mutarotatioti and the terminal rotations obtained on standing a t low temperature have also been investigated (von Hippel and Wong, 1961). In agreement with the findings of Carpenter and Lovelace, these studies have shown, as might be expected, that the effect is not simply one of ionic strength, but varies enormously from one ionic species to anot,her. Thus, while small increments of (dciuni, barium, lithium, thiocyanate, bromide, etc., ions have large effects 011 the initial rates, other ions, particularly tetramethylammonium and acetate, have almost no effect at all. It seems reasonable to assume, in line with the discussion given earlier, that ions exert their effects by modifying the solvation of the gelatin polypeptide chains. The proline and hydroxyproline residues are fundamental in the processes
STRUCTURE O F COLLAGEN AND GELATIN
121
which have been described above. It is not simply that these residues “fit into” a left-handed helix as suggested by some authors. In our view, it is more reasonable to suppose that their geometry and rigidity, detailed in Section 11, establish and direct the left-handed configuration which is formed a t low temperature. The optical rotatory evidence suggests that elements of the poly-L-prolirie 11-type structure remain in the gelatin chain above the transition temperature, T,. It seems likely that this residual structure is due to the presence of a significant number of contiguous proline residues in the chain, a situation recurring frequently via the sequence Gly.Pro.Hypro.22 These regions may act as crystal nuclei for the growth of the poly-L-proline I1 helices. We may imagine, for definiteness, that these segments “lock-in” when the temperature is lowered below T,. As suggested earlier this may occur through the formation of water bridges between adjoining carbonyl oxygen atoms, the peptide segments neighboring the nuclei slowly crystallizing into the preordained poly-L-proline I1 helix through this type of hydrogen bond mechanism. It is possible that nucleation may also occur at the G1y.Pro.X sequences in the chain, although this situation seems less likely because of the increased number of bonds allowing free rotation. Additional supporting evidence for the type of nucleation proposed above comes from studies in which the enzyme collagenase was used to probe the configurational changes occurring during the gelatin -+ collagen-fold transition. As pointed out in Section 111, the requirement for activity of this enzyme is the peptidc sequence -1’ro.X.Gly.Pro-, with cleavage taking place between X and Gly. At temperatures above T,, von Hippel arid Harrington (1959) found all peptide linkages in ichthyocol gelatin cleaved by the enzyme to belong to a single class in that the reaction obeyed simple first-order kinetics over several half-lives. When the gelatin solution is cooled below the transition temperature, the kinetics become complex and analysis suggests that the potentially cleavable bonds are distributed among two classes with about 20% of the total undergoing cleavage at a rate nearly tenfold that of the remaining bonds. Moreover, the complex kinetics attain their final form within 1 hr after lowering the gelatin temperature (i.e., within the time required to complete an enzymatic reaction) in keeping with the proposal that the enzyme is sensing primarily the nucleation process. In view of the requirement for the peptide sequence Pro.X.Gly.Pr0.Y 22
Heyns and Legler (1960) have recently found t h a t pyrrolidine residues separated
by one or two non-imino residues can still generate a left-handed structural contribution t o optical rotation in single peptides. Thus the residue rotation of proline in the tripeptide carbobenzoxy-Gly.Gly.Pro-NHa is -217” whereas t h a t estimated for carbobeneoxy-Pro.Ala.Gly.Pro-NHais -272” and that for csrbobenzoxy-Pro.Ala.Gly.Hypro is -308”.
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HARRINGTON AND VON HIPPEL
by the enzyme, it seems reasonable to assume that the change in the form of the enzyme kinetics below T,,, results from a rapid, temperature-dependent alteration in the chain configuration in this region. The types of peptidc linkages cleaved can be divided into two general classes: those which result in a terminal G1y.Pro.Y sequence (amounting to about 80% of the total peptides formed) and the remaining 20% which have Gly.Pro.Hypro as the terminal sequence. The correspondence between the ratio of bonds cleaved in each reaction and that expected for these two classes of peptides indicates that below T , the sequence Pro.Hypro.Gly.Pro is cleaved much more readily than the sequence Pro.X.Gly.Pro.Y, and that this difference in rate is related to the nucleation or “lock-in” phenomenon about the Pro.Hypro elements discussed above. We should note that the number of these nuclei would be relatively small compared to the total residues in a chain, so that significant changes in optical rotation would not be expected during the nucleation step. It is possible that some change occurs but that it is overshadowed by the mutarotation taking place during the time required for completion of an enzymatic reaction. In summary, tlhe following three-step mechanism for the reformation of the collagen-type structure in dilute gelatin solution, following cooling to temperatures below T,, has been proposed (von Hippel and Harrington, 1959; von Hippcl and Harrington, 19GO; Harrington and von Hippel, 1961). 1. An initial configurational change takes place in the pyrrolidine-rich portions of the gelatin chains, which “niicleat,es” the poly-L-prolinc II-type helix. This st,ep goes to completion rapidly arid is detected by the change from simple to complex collagenase kinetics. 2. The poly-L-proline I1 configurat,ion propagates outward from these nuclei along single gelatin chains. This process is responsible for the more rapid, concentration-independent portion of the mutarotation phenomenon. 3. The formation of the unique collagen-fold type structure along individual chains makes possible lateral chain association, which may be monitored hy the relatively slow changes in viscosity and light, scattering accompanying this step.
4. Properties of Gelatin Gels The physical properties of gelatin gels and their dependence on protein concentration, temperature, molecular weight, pH, and added reagents have been thoroughly reviewed by Ferry (1948a). We shall refer only briefly to supplementary work. Considerable insight into the mechanism of gelation is afforded by a study of the influence of chain weight on gel behavior. Below a chain weight of about 60,000, the rigidity of gelatin gels (shearing stress/shearing strain) which have been matured at low temperatures, increases markedly with
STRUCTURE OF COLLAGEN AND GELATIN
123
increasing molecular weight (Ferry and Eldridge, 1949; Saunders and Ward, 1958a). Above this critical size the rigidity appears to be essentially independent of molecular weight (Saunders and Ward, 1958b) and, in fact, may decrease somewhat (Stainsby, et al., 1954; Pouradier et al., 1954; Saunders and Ward, 1958b). Similarly, although Ferry observed the specific levorotation of mature gelatin gels to increase progressively over the molecular weight range 33-72 X lo3, it appears from the work of Saunders and Ward (1958) that [a], is unaffected by chain weight above this range. Melting behavior reveals, correspondingly, that the melting point is elevated appreciably with increasing chain weight below Mw = 70,000, whereas the relative change in melting temperature with molecular weight is distinctly reduced at higher Bwvalues (Ferry and Eldridge, 1949; Saunders and Ward, 1958a). Moreover, the melting point is substantially unchanged by variations in concentration a t high molecular weight (>70,000), but rises sharply with increasing concentration a t low chain weights (Ferry and Eldridge, 1949). Qualitatively, the mechanical behavior of gelatin gels resembles that of rubber. The damping of elastic response is small and there is a rapid response t o stress, analogous to that observed for rubberlike materials. On the molecular level, these properties are characteristic of network gels in which the interaction between chains occurs a t specific loci distributed at, intervals along each chain. In polymeric systems where chain-chain interaction can occur at every residue along the molecule, the resulting gel exhibits appreciable elastic damping, response to stress is slow and, moreover, gelation generally requires a careful balance between precipitation and solution. A number of recent studies on gelatin gels have supported and amplified the rubberlike model (Hirai, 1953; Saunders and Ward, 195813; Hastewell and Roscoe, 1956; Jopling, 1956) and are in general compatible with Ferry’s early postulate (1948a) that the loci available for cross-linking on each gelatin molecule are both specific in nature and limited in number. At low molecular weight it might be supposed that the number of these interaction sites would be too small t o form a stable network. When the chain length reaches a critical size, a relatively rigid lattice can be generated and the physical properties of the gel state should be less dependent on the length of each polypeptide chain. a. Dependence of Melting Points on Added Reagents. The melting points of gelatin gels exhibit only a small dependence on pH (Ferry) 1948a; Bello et al., 1956) but demonstrate a very marked dependence on the presence of certain salts, acids, and nonpolar substances. The effectiveness of salts in lowering the melting point has been found to be additive with respect to their ions, with highly hydrated cations such as Li+, Ca++, and Mg++ and large nonpolar or large, highly polarizable anions (Gordon and Ferry, 1946;
I24
HARRINGTON AND VON HIPPEL
Ferry, 1948a) bringing about the most dramatic effects. Thus sodium chloride, a t a concentration of 1 M , lowers the melting point of gelatin about (diiodosalicylate), 2.4"C, whereas lit~hium-2-hydroxy-3,5-diiodobenzoate TABLEXVI Melting Points of 6% Gelatin Gels Containing Salts" Salt None Sodium fluoride Sodium methanesulfonate Sodium chloride Sodium bromide Sodium nitrate Sodium thiocyanate Sodium iodide Sodium benzenesulfonate Sodium salicylate Sodium trimethylacetate Sodium chloroacetate Sodium dichloroacetate Sodium trichloroocetate Sodium dibromoacetate Sodium tribromoacetate Sodium diiodoacetate Sodium trifluoroacetate Sodium acetylt ryptophan Sodium maleate Sodium succinate Sodium fumarate Sodium acetylenedicarboxyltte Lithium chloride Lithium iodide Lithium salicylate Lithium diiodosalicylate Calcium chloride Magnesium chloride
Concentration (moles/liter) 1.0 1 .o 1 .o 1. 0
1 .o 1 .o 1.0
1.0 1 .o 1. 0 1 .o 1 .o 1.0 1.0 1.0 1.0 1.0 0.75 0. 5 0.25 0.5 0. 5 1.0 0.25 0.25 0.23 1 .o 1 .o
Melting point ("C) 30.4 34.5 31.5 28.0 22.8 22.3 16.0 10.0 15.5 No gel 26.5 26.6 19.6 12.7 16.3 No gel 12.3 24.1 13.7 33.7 32.3 31.5 19.4 25.4 26.7 20.5 No gel 16.1 22.9
From Bello et n l . (1956). Reproduced with kind permission of the American Chemical Society.
atl an cquivalent concentration, lowers the melting point by 120°C (Rello et aE., 1956). Table XVI, taken from the work of Bello, Ricse, and Vinograd, lists the melting points of gelatin gels containing a wide variety of salts. I n general the order of effectiveness of cations and anions follows a Hofmeister series (I
STRUCTURE OF COLLAGEN AND GELATIN
125
High concentrations of the competitive hydrogen-bonding reagents urea and guanidinium chloride also show strong depressant effects on the melting temperature, with guanidinium ion being about twice as effective as urea. On the other hand, little change in melting point is observed in the presence of guanidinium sulfate, the elevating effect of the sulfate anion counteracting the depressing effect of the guanidinium ion. I n a comparative study of the gelation of various gelatins which had been modified chemically so as to mask polar groups, Bello et al. (1956) have shown that the striking alterations in melting point in the presence of electrolytes are essentially unaffected by these changes, and therefore cannot depend on ion binding at the polar groups. They suggest binding at the peptide linkages as t hc most likely explanation. 1). DPpendence of Optical Rotation on Added .Reagents. In general the effect of various electrolytes on the optical rotation of gelatin gels closely parallels their effect on the melting point. Most salts lower the specific levorotation appreciably, with a corresponding loss in gel rigidity. The detailed study of Carpenter and Lovelace (Carpenter, 1927; Carpenter and Kucera, 1931; Carpenter and Lovelace, 1935a,b, 1936, 1937) remains the classic work in this field and demonstrates that the change in optical rotation of gelatin gels (at 0.5"C) follows a Hofmeister series. Comparing the salts of potassium, Carpenter and Lovelace found the following order in the effectiveness of the anions: CNS- > I- > c103- > NO,- > Br- > C1- > CHICOO- > CH3CH200- > HCOO-. The similarity of this series with that laid out by Bello, Riese, and Vinograd (see Table XVI) for the melting point of the gels clearly indicates the close relationship between the optical rotatory and melting phenomena. This view is supported by iiivestigations on numerous other reagents; those substances which have a strong effect on rigidity and melting point show a qualitatively similar response in optical rotatory properties (Katz and Wienhoven, 1933; Ferry, 1948a; Bello et al., 1956). c. The Mechanism of Gelation. A number of workers have investigated the possibility that the polar side-chain groups plily a significant role in the gelation phenomenon. Grabar and Morel (1950) reported inhibition of gelation when approximately 45 % of the guaiiidino groups of arginine arc destroyed by alkaline hypobromite, and suggested that these groups are responsible for gelation. Later work by Davis (1957) supported this view but indicated that gelation was only inhibited when the amount of hypobromite is sufficient to destroy virtually all of the guanidino groups. The interpretation of these experiments has recently been contested b y Bello (1959) who finds that hypobromite reduces the gelatin chain weight appreciably, suggesting that rigidity is depressed through inability to form a stable gel network. Moreover, chemically modified gelatins in which thc
126
HARRINGTON AND VON HIPPEL
polar groups are either masked or altered exhibit only minor changes in gelling properties. Gelatins with amino and hydroxyl groups acetylated, carboxyl groups esterified, and guanidino groups either nitrated or increased in number have essentially the same melting points, rigidities, and solution viscosities as unmodified gelatins (Bello et al., 1956; Kenchington, 1958). Since the side-chain groupings do not appear to play a fundamental role in the gelation process, the mechanism of gelation must involve, primarily, groups along the polypeptide backbone. The probable involvement of peptide groupings is also indicated by work on the gelling properties of biuret-gelatin complexes (Bello and Vinograd, 1958). At peptide to copper ratios as high as 17, gelation was completely suppressed a t O'C, but could be restored by destroying the biuret complex a t low pH. As a result of thew studies Bello and Vinograd have proposed that blocking of the peptide groups may interfere with the formation of necessary cross-linkages at thesc sites or, alternatively, that the copper complex may interferc with the formation of a required intramolecular configuration. Recently it has been found that formation of the biuret complex also suppresses mutarotation almost completely, while acidification (and coiisequent destruction of the complex) reactivates the mutarotation process (von Hippel and Wong, 1961). It was inferred from these results that chelation of the cupric ion across the peptide bond inhibits the development of the poly-L-proline 11-type helix by restraining adjacent residues in sterically unfavorable configurations. Many of the physical properties of gelatin gels are consistent with thc assumption that the formation and structural integritiy of the gel network depends on the establishment of a specific configurational pattern along segments of the individual gelatin chains. This point of view was firstj proposed by Hermann and Gerngross (1932). As a result of their X-ray diffraction studies of gelatin gels they suggested that the nctwork is formed through crystallization of certain regions of the gelatin chain, these segments being associated 1:tterally to form rrystalline bundles of chains, or xystallites. Ferry (19484 latcr argued that the chains might first associate in pairs rather than bundles, with further association of paired chains leading to the formation of crystallites. He estimated that as few as 5 or 6 loci per gelatin chain would be sufficient to form a rigid network. The proposal that gelation proceeds through the formation of crystallites is also supported by the light-scattering results of Boedtker and Doty (1954) and by the wellknown decrease in volume and evolution of heat characteristic of crystallization processes (Flory and Garrett, 1958; Flory and Weaver, 1960). In the present review, as indicated earlier, we have assumed that the association loci of the gelatin chains are segments of the chain arranged in
STRUCTURE OF COLLAGEN AND OEILATTN
187
the poly-L-proline I1 helix configuration. The somewhat broad meltingout profile of the gelatin gel would result from the lack of continuity in order along the chain. The close relationship between the melting point of the gel and the temperature at which the high levorotation is attenuated is understandable, since both phenomena depend on the integrity of the intrachain helices. This close relationship is also demonstrated by the analogous effect of salts on the melting point and on optical rotation. Destruction of the poly-L-proline I1 helices through the action of salts, by way of a mechanism such as that proposed earlier, would destroy the loci necessary for maintenance of the gel network. Since the association between gelatin chains leads to partial restoration of the collagen structure, it will be evident that close packing of the chain segments allows the type of interchain hydrogen bonding found in collagen. I n fact, if the proper conditions of temperature and concentration are employed, the gross macromolecular properties of collagen can also be regenerated. Veis and Cohen (1960) found that cooling a fractionated 7 % bull hide corium gelatin at 4°C for 20 hr, followed by a short heating step a t 40”C, resulted in the precipitation of fibrils with the cross-striated pattern typical of native collagen. The fibril width was variable, but electron micrographs revealed typical -640A spacings along the longitudinal axis. Rice (1960) has recently utilized the technique of Hall and Doty (1958) for viewing individual molecules in the electron microscope, and has obtained rods on spraying cold gelatin which have morphological properties similar to those of native collagen. Calfskin collagen solutions a t concentrations between 0.2 and 0.4 % were heated 19 min a t 70°C and sprayed a t elevated temperatures onto freshly cleaved mica surfaces. After shadowing with platinium, electron micrographs of these preparations showed only globules. When the heated solutions were stored a t 5°C for several hours before spraying, regenerated rods were observed. Although these varied somewhat in length, many showed the 2800-3000 A length and 15 A diameter charxteristic of the native collagen molecule. Typical SLS-type fibrous aggregates were also obtained using the “renatured” collagen. ACKNOWLEDGMENTS Preparation of this article was supported by research grants (A-4349) and (A-3412) from the National Institutes of Health, United States Public Health Service One of us (P. H. von Hippel) is indebted t o the Public Health Service for a Senior Research Fellowship (SF-360). Thanks are also due Miss S. Himmelfarb and Mrs. M. Backer for help in t h e final preparation of the manuscript, and t o Mr. H. Walton for construction of the wire models of poly-L-proline.
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Randall, J. T., Fraser, R. D. B., and North, A. C. T. (195313). Proc. Roy SOC.B141, 62. Randall, J. T., Booth, F., Burge, R. E., Jackson, S. P., and Kelly, F. C. (1955). In “Fibrous Proteins and Their Biological Significance.” Symposia SOC.ExptZ. B i d . 9, 127. Rice, R. V. (1960). Proc. Natl. Acad. Sci. U.S. 46, 1187. Rich, A. Personal communication. Rich, A., and Crick, F. H. C. (1955). Nature 176, 915. Rich, A., and Crick, F. H. C. (1958). In “Recent Advances in Gelatin and Clue Research” (G. Stainsby, ed.), p. 20. Pergamon Press, London. Rich, A., and Crick, F. H. C. (1959). Personal communication from A. Rich. Rich, A , , and Crick, F. H. C. (1961). J . Mol. Biol. 3, 71. Robb-Smith, A. H. T. (1957). I n “Connective Tissue.” Council Intern. Org. Med. Sci. Symposium (R. E. Tunbridge, ed.), p. 177. Blackwell, Oxford, England. Robb-Smith, A. H. T. (1958). I n “Recent Advances in Gelatin and Glue Research,” (G. Stainsby, ed.), p. 38. Pergamon Press, London. Robinson, C. (1953). I n “Nature and Structure of Collagen,” (J. T. Randall, ed.), p. 96. Academic Press, New York. Robinson, C., and Bott, M. J. (1951). Nature 168, 325. Rougvie, M. A., and Bear, R . S. (1953). J . A m . Leather Chemists Assoc. 48, 735. Sasisekharan, V. (1959a). Actu Cryst. 12, 897. Sasisekharan, V. (1959b). Actu Cryst. 12, 903. Sasisekharsn, V. (1961). In “Central Leather Research Institute Symposium on Collagen” (N. Ramanathan, ed.). Interscience, New York. I n press. Saunders, P. R., and Ward, A. G. (195%). In “Recent Advances in Gelatin and Glue Research” (G. Stainsby, ed.), p. 197. Pergamon Press, London. Saunders, P. R., and Ward, A. C. (195813). “Rheology of Elastomers,” p. 45. Pergamon Press, New York. Scatchard, G., Oncley, J. L., Williams, J. W . , and Brown, A. (1944). J . A m . Chem. Sac. 66, 1980. Schellman, C., and Schellman, J. A. (1958). Compt. rend. trav. lab. Carlsberg SO, 463. Schellman, J. A. (1955). Compt. rend. trav. Zab. CarZsberg 29, 230. Schellman, J. A. (1958). J . Phys. Chem. 62, 1485. Schellman, J. A , , and Schellman, C. (1961). J . Polymer Sci. 49, 129. Schmitt, F. 0. (1956). Proc. Am. Phil. Sac. 100, 476. Schmitt, F. 0. (1959). Revs. Modern Phys. 31, 349. Schmitt, F. O., and Gross, J. (1948). J . A m . Leather Chemists’ Assoc. 43, G58. Schmitt, F.O., Hall, C. E., and Jakus, M. A. (1942). J . CeZZular Comp. Physiol. 20, 11. Schmitt, F. O., Hall, C. E., and Jakus, M. A. (1945). J . Appl. Phys. 16, 263. Schmitt, F. 0.)Gross, J., and Highberger, J. H. (1953). Proc. Natl. Acad. Sci. U.S. 59, 459. Schmitt, F. O., Gross, J., and Highberger, J. H. (1955). In “Fibrous Proteins and Their Biological Significance.” Symposia SOC.Exptl. Biol. 9, 148. Schneider, F. (1940). Collegium, p. 97. Schroeder, W. A,, Honnen, L., and Green, F. C. (1953). Proc. N a t l . Acad. Sci. U.S. 39, 23. Schroeder, W. A., Kay, L. M., LeGette, J., Honnen, L . , and Green, F. C. (1954). J . Am. Chem. SOC.76,3556. Schrohenloher, R. E., Ogle, J. I)., and Logan, M. A . (1959). J . Rial. Chem. 234, 58.
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THE PROTEINS OF THE EXOCRINE PANCREAS By P. DESNUELLE and M. ROVERY Labaratoire de Chirnie Biologique, Faculti der Sciences, Marseille, France
I. Introduction.. .......................................................... 11. Biosynthesis of Pancreatic Enzymes. .................................... A. General Techniques for Determining Pancreatic Enzymes . . . . . . . . . . . . . B. Biosynthesis and Localization of Enzymes in Acinar Cells 111. Recent Advances in the Chemical Characterization of Some Proteins of Exo.............................. enclature. .................... B. Bovine Chymotrypsinogen A . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bovine Chymotrypsinogen B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Porcine Chymotrypsinogen A , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Bovine Trypsinogen and Trypsin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Porcine Trypsinogen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Bovine Procarboxypeptidase A and Carboxypeptidase A . . . . . . . H. Bovine and Porcine Carboxypeptidases B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Porcine Lipase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Bovine Ribonuclease., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Sheep and Mouse Ribonucleases.. . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
139 141 141 148 153 153 154 163 165 167 171 173 175 176 187 191
I. INTRODUCTION^ A brilliant success of modern biochemistry has been to show that all or almost all proteins of bovine pancreatic juice are enzymes (1).It may be said in a cursory way that this juice is a solution of enzymes in bicarbonate and that its composition is strictly utilitarian. Bicarbonate is present in order to neutralize hydrochloric acid coming from the stomach and to keep enzymes in solution. Enzymes are present for digesting alimentary products in the intestine. On the other hand, pancreatic juice seems to have been created for the protein chemist’s delight, since its proteins are biologically active, relatively simple, and endowed with unusual properties. Intraluminar digestion consists of a series of hydrolyses converting large molecules with specific structures into smaIler molecules able to force their way into the intestinal mucosa. In many species, pancreatic juice contains 1 The following abbreviations are used in the text: DEAE-cellulose, diethylaminoethyl-cellulose; CM-cellulose, carboxymethyl-cellulose; DFP, diisopropylphosphofluoridate; ATEE, acetyl-L-tyrosine ethyl ester; BAEE, benzoyl-L-arginine ethyl ester; Tris, tris(hydroxymethy1)aminomethane.
139
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one or several hydrolases for each main component of the diet: a series of proteolytic enzymes for proteins, a lipase, a phospholipase, and a cholesterolesterase for lipids, an amylase for polysaccharides, a ribonuclease and a deoxyribonuclease for nucleic acids. The fact that pancreatic enzymes are exclusively hydrolases interests protein chemists as well as enzymologists. Most oxidoreductive enzymes consist of a protein carrier and a small coenzyme to which the largest part of the catalytic effect may be attributed. On the contrary, many hydrolases merely consist of a holoprotein in which the active site is formed by a tridimensional and specific arrangement of amino acids residues. One of the most challenging problems of molecular biology is to find out a correlation between catalytic activity and protein structure. Even when a metal ion is involved in the catalytic activity of hydrolases or in their stability, these ions appear to be bound in a specific way to some groups of the protein molecule and their action can be discussed in terms of protein structure. The study of pancreatic enzymes at the molecular level has been facilitated by two circumstances: (a) pancreatic juice and pancreas extracts are simpler than many other biological fluids; (b) most of their proteins have a relatively low molecular weight. The proteins behave well during fractionation procedures based on molecular kinetics and some hope exists of determining their structures with the available techniques of protein chemistry. The enzymatic activity of pancreatic proteins may be regarded as an additional attraction and a help in tracing these proteins during purification and in demonstrating their homogeneity. The external secretion of the pancreas has other connections with protein chemistry. In the first place, it is known to be stimulated, not only by nerves and cholinergic drugs, but also by two hormones, secretin and pancreozymin. Secretin, discovered in Ivy’s laboratory by Hammarsten and Agren, has been purified to a considerable extent from hog intestine (2). I t mainly promotes a flow of water and mineral salts into the secretory ducts. The enzyme content of pancreatic juice seems, however, to be controlled by nervous stimulation and by pancreozymin. This second hormone, discovered by Harper and Raper in 1943, is not yet fully characterized (3). But it may be easily understood that the mobilization of enzyme proteins stored behind the membranes of zymogen granules (to be discussed later) and the passage of water molecules through the walls of the secretory ducts are two distinct processes requiring two different types of stimulation. Although the whole problem of the stimulation of the exocrine pancreas is not discussed here for obvious reasons, it will be pointed out that it induces a relatively large protein output to which must correspond a very active protein biosynthesis. Cannulated steer pancreas for instance excretes
PROTEINS OF EXOCRINE PANCREAS
141
an average of 1 gm of protein per hour (4).Mouse pancreas, which represents only 0..5-0.6% of the total body weight of the animal, concentrates about 10% of injected radioactive valine ( 5 ) . Again, the activity of pancreatic enzymes and the relative ease with which they can be fractionated facilitate an accurate study of their biosynthesis in intact animals and their possible variations with external factors. Finally, the presence in pancreas acinar cells of cytoplasmic components and enzymes able to hydrolyze them requires some protective devices which can be partly elucidated by the techniques of protein chemistry. The first part of this review will be devoted to enzyme biosynthesis by pancreas and the second part to chemical characterization of certain pancreatic enzymes. Some of these enzymes have already been comprehensively discussed in recent volumes of this series (6, 7).
11. RIOSYNTHESIS OF PANCREATIC ENZYMES A . General Techniques for Determining Pancreatic flnxymes The intrrest of experiments designed to investigate enzyme biosynthesis by pancreas strongly depends upon the accuracy of available techniques for the determination of enzymes in such complex mixtures as pancreatic juice, pancreas homogenates, or lysates of zymogen granules. Significant technical advances have been made recently in this direction. They have been facilitated by previous investigations carried out in vilro with purified enzymes of bovine pancreas. 1. Chromatography
I n 1953, Hirs (8) found that well separated peaks of chymotrypsinogen A and ribonuclease A could be directly obtained by chromatographing I
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proteins should present themselves in an order of decreasing isoelectric points. This is actually the case except for ribonuclease which is the last protein to be eluted from Amberlite IRC-50 under the conditions of the experiment, although it is isoelectric at a lower pH than trypsinogen and chymotrypsinogen A. The data of Fig. 1 appear to be of fundamental importance for a number of reasons. After two chromatographic steps, 90% of the total proteins of bovine juice has been fractionated in a series of discrete peaks. Almost all peaks can be associated with known enzymatic activities. Thus, a qualitative picture of the enzymatic content of the juice is obtained at once. It
I
-
ChTg B
+Cotion
exchange+Anion -Effluent
exchangevolumes-
FIG.1. Chromatography of bovine pancreatic juice on DEAE-cellulose (anionic proteins) and Amberlite IRC-50 (cationic proteins) (1). RNAase, ribonuclease; ChTg-a, chymotrypsinogen A; Tg, trypsinogen ; ProCp-B and Cp-B, procarboxypeptidase B and carboxypeptidase B; DNAase, deoxyribonuclease; ProCp-A, procarboxypeptidase A.
will be interesting to locate on the diagram somewhat ill-defined pancreatic activities named elastase (9, lo), insulinase (lo), protaminase (ll),pankrin (12), and “esterases” by applying their specific tests to each fraction. It should be possible in t,his way to see whether or not the corresponding enzymes really exist. On the other hand, quantitative information about the relative and absolute proportions of pancreatic enzymes can be obtained by determining the peak areas. But, for such information to be correct, the enzymes must be eluted from the column completely. They should also have been obtained previously in a pure form so that their extinction coefficients are known. Finally, the peaks should be pure. All these rather drastic requirements seem to be fulfilled in the case of bovine pancreatic juice. A quantitative balance sheet (1) can therefore be established with the following results:
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PROTEINS OF EXOCRINE PANCREAS
chymotrypsinogen A, 16 % of the proteins of the juice; chymotrypsinogen B, 16 %; trypsinogen, 14 %; procarboxypeptidase A, 19 %; procarboxypeptidase B, 7 %; ribonuclease, 2.4 %; deoxyribonuclease, 1.4 %; amylase, less than 2 %; lipase, very low; unidentified, 10 %. I n a more recent work (4) , it has been found by the same techniques that the proportions of the cationic proteins of bovine juice remain fairly constant during a 5-hr period of secretion and that they are 15% for chymotrypsinogen A, 16% for trypsinogen, and 3.3% for ribonuclease (weight ratio, 5 : 5 :1; molar ratio, 3:3:1).
I Tg
0.750
j
Chtg
j
Pro CP-A I
0.500
0,250
0
:
RNAose
I
-Cotions
0
/
,
Anions 44
I
6
8
FIG.2. Chromatographic diagram of porcine pancreatic juice (13, 14). Abbreviations used for enzyme names are the same as in Fig. 1. The anionic proteins are fractionated on a DEAE-cellulose column equilibrated with 0.005 M phosphate, pH 8.0 and eluted a t the same p H with a concentration gradient. The cationic proteins are fractionated on a CM-cellulose column by stepwise elution with buffers of increasing pH's. Ordinates, optical density of the fractions at 280 mp. Abscissas (on the right diagram), volume of eluate expressed in number of interstitial volumes of the column.
These results give, for the first time, reliable information about the general composition of bovine juice. Its content of proteolytic enzymes is strikingly high (about 72 % of the total proteins). Chymotrypsinogen B, which was formerly considered as a minor constituent, is very abundant. On the other hand, bovine juice is quite poor in amylase and lipase. Figure 2 gives the diagram obtained with porcine juice collected by a permanent fistula (13, 14). I n this case, Amberlite IRC-50 has been replaced by CM-cellulo,se for the fractionation of the cationic proteins. But the other conditions are the same as in the preceding experiment. The first remark suggested by Fig. 2 is that porcine juice contains relatively large amounts of amylase and lipase. Pig amylase (15) and pig lipase (16, 17) have already been purified. The difference existing in this
144
P. DESNUELLE AND M. ltOVMltY
respect between pig and cattle juices is quite striking. As stated above, the main components of bovine juice are proteolytic enzymes. The porcine secretion is much more diversified. The physiological significance of this difference is still unknown. Since preliminary experiments have shown that dog (see below), rat, and human juices also contain large amounts of amylase and lipase, it may be postulated that the bovine secretion represents a special case associated with rumination. I n other words, fats and carbohydrates are perhaps hydrolyzed by the bacteria of the rumen before reaching the intestine. Figure 2 shows further that very large amounts of promrboxypeptidasm
-I
PlOCpA
5,. bl
075
Amylase
Lipase
DNAase
Fro. 3. Protein and activity peaks obtained by chromatography of porcirio p:tiicretltic juice on DEAE-cellulose (13). The solid lines give the enzymatic activities of the fractions. The dotted lines give the protein background taken from the corresponding zones of Fig. 2.
A and B are present in porcine juice. However, thc order of emergence of these precursors is not the same as for bovine juice. Moreover, a single chymotrypsinogen peak is present instead of two as in bovine juice. This peak is found on the cationic side, although the isoelectric point of thc corresponding protein is 7.2 (to be discussed later). It will be referred to as porcine chymotrypsinogen A. The possible existence of a second porcine chymotrypsinogen is discussed later. Finally, the order of emergence of chymotrypsinogen A and trypsinogen is not the same here as for bovine j uice , Figure 3 gives the correlation existing between protein peaks as determined by ultraviolet absorption and the enzymatic activities of the fractions (13). The correlation is good. A single activity is found under most of the peaks but ribonucleolytic activity is located, not only under the
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PROTEINS OF EXOCRINE PANCREAS
ribonuclease peak, but also in other parts of the diagram. Furthermore, utilization of the diagram for quantitative purposes is rendered uncertain by two facts: (a) some porcine enzymes have not yet been purified. Except in the cases of amylase, chymotrypsinogen A, and trypsinogen (to be discussed later), their extinction coefficients are not known. (b) The specific activities found under some peaks are lower than normal. The porcine
t A
0.750
10
n
FIQ.4. Chromatography of dog pancreatic juice on DEAE-cellulose (13, 14).The column is equilibrated with 0.005 M phosphate, pH 8.0 and eluted by a concentration gradient of phosphate indicated in the figure by a straight line. (1) Unfractionated cationic proteins; (2) amylase; (3) lipase; (4)deoxyribonuclease; ( 5 ) anionic chymotrypsinogen; (6), (9), and (10) carboxypeptidase A and its precursor; (7) and ( 8 ) carboxypeptidase B and its precursor. Ordinates on the left, optical density of the fractions at 280 mp. Ordinates on the right, molarity of the phosphate. Abscissas, volume of eluate expressed in number of interstitial volumes of the column.
enzymes are apparently more labile than their bovine analogs during chromatography on DEAE-cellulose at pH 8.0. Figure 4 gives the result of a single and preliminary experiment with dog juice (13, 14). Its main interest is to show that a “map” of the enzymatic equipment of the pancreas of various species can be obtained by chromatography in a relatively short time. Similar chromatographic techniques have been recently used for the fractionation of liver extracts (18). About sixteen peaks with known activities have been identified. But the liver enzymes are very numerous indeed, and their systematic fractionation represents a more difficult problem.
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Thus it seems that, at least for the time being, a complete and quantitative diagram can only be obtained with bovine juice. But chromatography gives a very good opportunity of drawing qualitative maps of the enzymatic
0
25
10
20
hr.
FIG.5. Kinetics of chymotrypsinogen(s) and trypsinogen activations in pancreatic juice and pancreas homogenates (22). Upper diagram: activation of chymotrypsinogen(s) in rat pancreas homogenate. Pancreas (1 gm) is homogenized 2 min a t 0°C and 2000 rpm in a Potter-Elvehjem apparatus with 9 ml of 0.25 M sucrose. One milliliter is diluted with 9 ml of 0.2 M phosphate buffer, pH 7.6 and incubated a t 0°C (white circles) or 35°C (black circles) with trypsin (1mg/100 mg of total protein). The chymotryptic activity is measured against acetyl-L-tyrosine ethyl ester. Abscissas, incubation time in minutes. Ordinates, specific activity of chymotrypsin (~-equivalents/min/mgof protein). Lower diagram: activation of trypsinogen in pig pancreatic juice. A lyophilized sample of juice is dissolved in a p H 7.9 buffer 0.005 M in Tris, 0.04 M in NaC1, and 0.02 M in CaClz , The solution (25 mg of protein/100 ml) is incubated with trypsin (4 mg/100mg of protein). The tryptic activity is measured against benzoyl-L-arginine ethyl ester. The activity of added trypsin is deducted from each result. Abscissas, incubation time in hours. Ordinates, specific activity of trypsin &-equivalents/min/mg of protein). Temperature: 0°C.
pattern of other species. These maps give valuable information for further purification. Chromatography is also extensively used for isolating enzymes from juice, pancreas, and its subcellular fractions (see later). These isolations are easier than complete chromatography, since the most suitable technique can be selected in each special case.
PROTEINS O F EXOCRINE PANCREAS
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2. Direct Determination of Enzymatic Activities
When quantitative chromatography gives inconclusive results, the general composition of the mixture can sometimes be ascertained by direct determination of enzymatic activities. If the specific activities of the pure enzymes are known, an estimation of their absolute proportions (grams of enzyme in 100 gm of total protein) can be made. In the reverse case, possible variations in the enzymatic pattern of a given species can at least be detected by determining activities for a certain volume of juice or for a certain weight of pancreas. The main difficulty of course is in finding satisfactory techniques for measuring enzymatic activities in complex mixtures. For obvious reasons, this latter problem will not be fully discussed here.
FIG.6. Activation of chymotrypsinogen(s) and trypsinogen as a function of added trypsin (22). On the left: chymotrypsinogen(s) activation in pig juice. Incubation time, 120 min. The other conditions are the same as in Fig. 5 . On the right: trypsinogen activation in pig juice. Incubation time, 18 hr. Abscissas, milligrams of added trypsin per 100 mg of total protein. Ordinates, specific activity (p-equivalents/ min/mg of protein) after deduction of the activity of added trypsin.
But an important point is to realize that: (a) comparisons at the molecular level cannot be established between species, since the same enzyme formed by various species may have different specific activities and require different cofactors. (b) Very large errors can be made, and actually have been made in the past, by using poor techniques for the determination of enzyme activity. At the present time, directly active enzymes such as amylase (19, 20), lipase (16), and ribonuclease (21) are measured with a reasonable degree of accuracy. Good techniques and specific substrates are also available for chymotrypsin, trypsin, carboxypeptidases A and B. But the special problem here is that proteolytic enzymes must be activated and that the activation step must be carefully controlled in complex mixtures (Figs. 5 and 6). Under the conditions selected (22), a well-defined and reproducible activity plateau is reached in Fig. 5 for chymotrypsin and trypsin. The height
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P. 1)ESNUELLE AND M. ROVERY
of this plateau is proportional to the amounts of homogenate or juice used. Figure 6 shows that there exists a lower limit for added trypsin below which trypsinogen activation does not start and chymotrypsinogen activation is very slow. This limit probably reflects the existence of trypsin inhibitors and may give an interesting expression of the stability of pig juice. When full activation is obtained, the amount of added trypsin may be varied within large limits without any detectable influence on the final activity values. Since the specific activities of pure porcine amylase (23), lipase (16), and trypsinogen (Section 111,F) are known, approximate values for the percentage of the three enzymes in pig pancreatic juice can be estimated as stated above. These values are respectively 7.5, 2.5, and 24% of the total proteins. By taking into account the specific activity of pure pig chymotrypsinogen A (Section 111, D), a value of 14% is found for this class of precursors. However, the value is preliminary since the specific activity and the amount of the second rhymotrypsinogen of pig are not yet known.
B. Biosynthesis and Localization of Enzymes in Acinar Cells The acinar cells of pancreas synthesize, not only structural proteins and enzymes for their own metabolism, but also exocrine or “exportable” enzymes which are carried into the duodenum for digestive purposes. To this dual function corresponds a rather complicated system involving, besides biosynthesis itself, segregation, transport, storage, and final excretion. 1. Histological Observations
It is generally assumed that protein biosynthesis occurs in or on small rihonucleoprotein particles named ribosomes. The rough-surfaced regions of the endoplasmic reticulum of pancreas acinar cells are studded with ribosomes (24). After disruption of the cell structure by homogenization, the endoplasmic reticulum is converted into microsomal vesicles which contain the molecules formerly present in the reticulum and to which the ribosomes are still bound. These ribosomes can be spun down as a separate fraction when the microsomes are dissolved by sodium deoxycholate (25). On the othcr hand, when guinea pigs are starved and killed 1 hr after feeding, the cavities (cisternae) of the endoplasmic reticulum of acinar cells contain granules and the microsomal fraction is more dense. When the starved animals are killed before feeding, the cavities are empty and the microsomal fraction is lighter. It is therefore tempting t o postulate that the intracisternal granules are formed by enzymes which have just been synthesized under food stimulation (25-27). Finally, it was already noted by Heidenhain (28) in 1875 that pancreatic acinar cells contained relatively large granules which occupied a large part
PROTEINS OF EXOCRINE PANCREAS
149
of the cytoplasm during starvation, but became fewer in number after stimulation of the exocrine secretion. These granules appeared to act as enzyme “stores” and were called zymogen granules. More recently, beautiful electron micrographs have confirmed that zymogen granules are associated with the exocrine secretion of pancreas. They are seen to coalesce between themselves and with the walls of the secretory ducts, and thus to discharge their content into the duct lumen within the limits of a closed system (27). On these observations is based the Palade and Siekevitz theory according to which pancreatic enzymes are synthesized in ribosomes, segregated into “exportable” and “nonexportable” enzymes in the reticulum cisternae and the Golgi apparatus. The exportable enzymes are then stored in zymogen granules until the next stimulation induces them to pass into the juice. It will be of interest to see to what extent this theory can be confirmed by chemical techniques. 2. Chemical Observations
a. Site of Biosynthesis and Localization. When C14-labeled amino acids are injected into animals, the trichloroacetic acid insoluble proteins of the pancreas rapidly incorporate radioactivity (29, 30). This observation has been confirmed and considerably extended by Morris and Dickman (5) and by Siekevitz and Palade (31, 32). The first authors injected C14-valine into mice and counted radioactivity in ribonuclease A separated in an apparently pure form from some subcellular fractions and supernatant according to the chromatographic technique of Hirs et al. (33). They found that radioactivity was already very high in microsomal ribonuclease 5 min after injection, that it reached a maximum in 15 min, and then declined. The rate of radioactivity incorporation was lower for supernatant and zymogen granules and still lower for nuclei. After 120 min, the highest radioactivity was found in zymogen granules. Similar results were obtained b y Siekevitz and Palade when guinea pigs received Cl4-leucine. After homogenization of the pancreas in 0.88 M sucrose, the zymogen granules were carefully separated from mitochondria by centrifugation in a discontinuous density gradient (26). The microsoma1 fraction was further fractionated into ribosomes, intracisternal granules, microsomal content, and two postmicrosomal fractions. The proteins of each fraction were separated into a n acid-soluble part which was considered as containing most of the exportable enzymes and an acidinsoluble part. Finally, chymotrypsinogen A was isolated from the acidsoluble part by chromatography on Amberlite IRC-50 (8). Radioactivity was determined in each fraction and each sample of chymotrypsinogen A. To restrict the discussion to chymotrypsinogen, radioactivity was highest
150
P. DESNUELLE AND M. ROVERY
after 1-3 miri in the samples prepared from ribosomes and then in the samples from intracisternal granules. It began to appear in the zymogen granules and the supernatant after 3 min and became highest in the zymogen granules after 45 min. The real significance of the radioactivity found in the supernatant cannot be estimated since the number of subcellular particles which were broken or lysed during homogenization and further treatment is unknown. With this restriction, the results were fully compatible with the assumption that exocrine enzymes are produced a t a very high rate in ribosomes, are packed together in intracisternal granules, and finally are stored in zymogen granules over a certain time. b. Correlation between Zymogen Granules and External Secretion. When zymogen granules are suspended in water or in a solution more alkaline than pH 7.2, they lyse (34) and their content may be subjected to chromatography in the manner described earlier for pancreatic juice. The diagrams obtained with the lysates of bovine granules and with bovine juice are qualitatively and quantitatively very similar (35-37). The same enzymes are found in nearly the same proportions. This very interesting similarity strongly suggests that the enzymes present in the juice come directly from the granules and it is therefore fully compatible with the view that exocrine enzymes are stored in the granules. But it must be recalled here that the main components of bovine juice are proteolytic enzymes and that intragranular localization of exocrine enzymes has only heen proved for this group so far. Deoxyribonuclease and ribonuclease are also present in zymogen granules. A special investigation has even been carried out to show that the granules contain ribonucleases A and B (37). But] the amounts of the nucleolytic enzymes are too low in bovine juice for an accurate quantitative comparison to be established and nothing is known so far for amylase and lipase. It would be interesting in this respect to isolate porcine zymogen granules and see how much lipase and amylase they contain. The problem of the real extent of intragranular localization of pancreatic mzymes is a rather academic one, since it is fairly certain that enzymes exist outside the granules. Some convincing arguments have been prewilted suggesting that ribonuclease, for instance, is not strictly localized. The enzyme extracted from mouse pancreas supernatant is sometimes more radioactive than samples extracted from granules and the excess of radioactivity of the first cannot be attributed to unsedimented microsomes ( 5 ) . Some “free” ribonuclease in the cytoplasm of guinea pig (26) and cattle (38) acinar cells seems to be able to bind a fluorescent antibody. On the other hand, proteolytic activity, as measured against casein after an uncontrolled activation, appears to be more strictly localized inside dog granules than amylase and lipase (34). The same is likely to be true for
PROTEINS OF EXOCRINE PANCREAS
151
amylase when compared with ribonuclease (39, 40). Finally, whcn mouse granules are mobilized by pilocarpine stimulation, the loss in the whole homogenate is 55 % for amylase and only 33 % for ribonucleaee (5). But the important problem which can be solved quickly by existing and reliable techniques is whether or not all exocrine enzymes are localized inside granules. Keller and Cohen (36) also subjected to chromatography acidic extracts of cattle pancreatic microsomes and ribosomes. In microsomes they found the expected amounts of all enzymes which are known to be stable in acid, viz., chymotrypsinogen A, trypsinogen, deoxyribonuclease, and ribonuclease. The amounts of chymotrypsinogen B were abnormally low and ribonuclease B was perhaps not present. The results concerning ribosomes were made somewhat uncertain by the ability of these particles to incorporate proteins from the medium. Nevertheless, a series of characteristic enzymes could be isolated from what appears to be the very site of their biosynthesis. 3. Biological Significance of Enzyme Localization
When a cell synthesizes an enzyme able, as pancreatic enzymes are, to hydrolyze its own constituents, some kind of protection must be provided. The first assumption is that the cell contains inhibitors which afford a temporary protection (41) until the enzyme is evacuated. The second is that the cell at first synthesizes inactive molecules which are activated later in the intestinal tract. The third is that digestive enzymes are not mixed in situ with cell constituents but separated from them b y impervious membranes. The existence of zymogen granules in pancreatic acinar cells is consistent with the third assumption. Enzymes would be synthesized and localized inside granules in order to provide, not only a possibility of storage between two secretions, but also a protection for the cellular constituents. I n other words, zymogen granules would be, at the same time, a storehouse and a jail. But the general significance of this assumption strongly depends upon the extent of intragranular localization for each individual enzyme. This extent is not yet known with absolute certainty. On the other hand, even if localization of proteolytic enzymes were complete these enzymes could be expected to digest the other enzymatic proteins in the granules. An additional protection must therefore be provided against them. Pancreas contains a series of proteins able to inhibit active trypsin by a reversible and stoichiometric reaction (31). Rut, their exact role is still unclear and it is not known whether they are really present in the granules. Quite obviously, the most efficient protection against proteolytic enzymes is the one discovered 25 years ago b y Northrop, Kunitz, and
152
P. DESNUELLE AND M . ItOVERY
IIerriott (42)-their synthesis by pancreas in the form of inactive precursors which are activated later by a limited proteolysis induced b y trypsin (7, 43-45). It is very striking indeed that inactive precursors have only been identified so far in the case of proteolytic enzymes and that all known proteolytic enzymes of pancreas exist in the form of inactive precursors: chymotrypsinogen A (42), chymotrypsinogen B (46), trypsinogen (42), procarboxypetidase A (47, 48), and procarboxypeptidase B (49-53). This type of protection is not restricted to pancreas, but is used for instance for blood [prothrombin (54), plasminogen (55)]. It is also striking that all pancreatic precursors are activated by trypsin which represents the driving force of the whole system and for which a special activator named enterokinase is available in the duodenum (,56, 57).
4.Net Synthesis Several experiments have been performed in the past for measuring the rate a t which the normal enzyme content of pancreas is restored after depletion by stimulation of the external secretion. This type of study is of value only when enzymatic activities are determined by reliable techniques. On the other hand, it has the advantage of measuring a net protein synthesis instead of merely relying upon incorporation of radioactive amino acids. It will perhaps be useful in the future to reinvestigate the whole problem with improved techniques. Morris and Dickman (5) for instance have recently published interesting curves showing the variations of amylase and ribonuclease content in mouse pancreas after pilocarpine sttimulation. Activity decreases at first, as stated above, b y 55% for amylase and 83% for ribonuclease, and then increases. The rate of increase however is much higher for amylase (normal level after 10 hr) than for ribonuclease (normal level after 21 hr) . Moreover, amylase content rises well above the normal level to reach 200% after 20 hr. Another interesting problem is the study of the enzymatic content of the pancreas, not only as a function of time after stimulation, but also as a function of various external factors such as the composition of the diet, and the nutritional state of the animals. Several laboratories have already attempted to establish the existence of an “adjustment” of enzyme biosynthesis to the main component of the diet and to find out the mechanism by which the digestive process may influence enzyme biosynthesis (58-60). Pancreatic juice collected over an extended period of time by a permanent fistula may be considered an ideal material for studying the net synthesis of a series of well-defined proteins. As soon as radioactive proteins are present in zymogen granules they may appear in the juice after a certain delay, which depends upon the conditions of the next stimulation. When rats in which a continuous flow of juice is promoted by secretin or
PROTEINS OF EXOCRINE PANCREAS
153
carbamoyl choline receive CI4-glycineby intravenous injectlion,the proteins of the juice do not become radioactive during the first 50 min. Radioactivity appears later and becomes maximal after 90 min (61). In more recent and comprehensive investigations (4,62), S35-cystineor C14-arginine were injected into yearling steers bearing a permanent fistula and the juice was collected in 1-hr periods during 8 hr or more frequently just after the injection. The catioriic proteins (trypsinogen, chymotrypsinogen A, and riboiiuclease) of each sample were separated by chromatography and the radioact,ivity was counted in each peak. The main difficulties in this type of experiment is that the pool of the radioactive amino acids is not known, that only a part, of the enzymes probably appear in the juice, and that it has not yet been definitely established that all exocrine enzymes follow the same route from the ribosomes to the juice. However, interesting differences have been found between chymotrypsinogeii and trypsinogen, on one hand, and ribonuclease on the other. Both proteolytic precursors seem to be synthesized a t proportional rates from common intermediates and also transported a t proportional rates to the juice. I n contrast, radioactivity appears more slowly in ribonuclease after the injection of radioactive amino acids, and the specific radioactivity of this protein is definitely lower.
111. RECENTADVANCES I N THE CHEMICAL CHARACTERIZATION OF SOME lJROTEINS OF EXOCRINE IJANCREAS
A . Introductory Remarks Concerning Nomenclature It, is perhaps risefril to discuss briefly at the beginning of t'his chapter the nomenclature used for describing the structure of a protein molecule and the proteolytic enzymes found in the external secretion of pancreas. 1. Protein Structure
It is now generally agreed that proteins contain several types of bonds inducing several types of structures. The simplest, idea is to correlate each structure with the corresponding bond type. But the choice of the best correlation seems to be somewhat difficult. From a purely chemical point of view, the fundamental fact is that proteins contain covalent bonds which are split by rather drastic means and more labile, noncovaleiit bonds which may be split or a t least altered by denaturation alone. Two types of covalent structures exist, involving peptide bonds and disulfide bridges respect,ively. Both structures should be clearly differentiated since they are destroyed by quite different chemical treatments (hydrolysis for the first, oxidation or reduction for the second). They will be called for this reason, respectively, the primary and the secondary structures. Conversely, several inter- or intrachain, noncovalent
154
P. DESNUELLE AND M. R0VEH.Y
structures may be expected to exist. Since, in the present state of our knowledge, their common characteristic is their noncovalent nature, it seems permissible to use a single expression for both of them, namely tertiary structure. An important fact is that the tertiary structure of a protein may be easily modified either by denaturation or by alteration in any one of the covalent structures. 2. Nomenclature of the Proteolytic Enzymes of Pancreas
Northrop and co-workers (42) characterized in bovine pancreas a single chymotrypsinogen which gave rise by activation to a certain chymotrypsin. This chymotrypsin seemed to be converted later into other active products. It was named a-chymotrypsin in order to underline its apparently direct filiation with the precursor. The other active products were named @- and y-chymotrypsins. When Jacobsen (63) discovered that a-chymotrypsin was not the primary active compound arising during chymotrypsinogen activation, he called this compound ?r-chymotrypsin and used for its autolysis product the first available greek letter (B-chymotrypsin). Finally, when a second chymotrypsinogen was found in bovine pancreas (46), it was called chymotrypsinogen B and one of the corresponding enzymes, chymotrypsin B. The first precursor was then called a-chymotrypsinogen or chymotrypsinogen A. This kind of nomenclature needs revision. It is proposed to term chymotrypsinogen A the more cationic, and chymotrypsinogen B, the more anionic of the proteins present in bovine juice which, under well-specified conditions, give rise to an ATEE-splitting activity. This rule may be easily extended to other species. Furthermore, it is felt that the available information (7, 43, 44, 64) about the chemical events associated with chymotrypsinogen activation should be used in chymotrypsin nomenclature. Thus, r-chymotrypsin, arising by the breakdown of a single peptide bond, would become chymotrypsin A1 In the same way, 6- and a-chymotrypsins would become chymotrypsins A:, and Ad. Proteins of the a-family which are likely to be intermediates during the formation of the a-compound (65) would be named chymotrypsins Aa In order to avoid confusion, the classic and the new nomenclatures will be used simultaneously in this review.
.
.
B. Bovine Chymotrypsinogen A 1 . COOH-Terminal Residues
The study of the terminal residues of bovine chymotrypsinogen A is beset with unusual difficulties. The iTHz-terminal half-cystine cannot be found unless the protein is oxidized by performic acid (66). It is not split
PROTEINS OF EXOCRINE PANCREAS
155
by aminopeptidase even when 100 enzyme units are used per mole of protein (67). The COOH-terminal residue cannot be detected by the usual hydrazinolysis technique and it is unavailable to carboxypeptidase A in the native protein. After denaturation of the protein by urea (67), and after performic acid oxidation or reduction of S-S bridges (68), carboxypeptidase A splits large amounts of several amino acids. A careful analysis of the kinetic curves and the negative results obtained by hydrazinolysis suggest that asparagine is COOH-terminal. All these observations show how cautiously the results given by the so-called end-group techniques must be interpreted. Bovine chymotrypsinogen A is not, as believed earlier, a cyclic protein. It contains an “open” chain beginning with half-cystine and ending with asparagine. The existence of a single chain is confirmed by the fact that reduction of the S-S bridges does not induce any change in the molecular weight (52). Since the protein contains only one chain, the carboxypeptidase technique may give some information about the COOH-terminal sequence. The common practice in the use of this technique is to determine the order in which the amino acids are liberated and to draw kinetic curves for each amino acid. The ideal case is met when large differences exist between the rate of splitting of the bonds. Ambiguous results are sometimes obtained either when all bonds are split rapidly or one bond near the carboxyl end is split much more slowly than the following ones, or, finally, when a given residue is present more than once in the available sequence. As already stated above, denatured or reduced chymotrypsinogen A in the presence of carboxypeptidase liberates many amino acids in large amounts. More than 1 mole of alanine is liberated from each mole of chymotrypsinogen. The sequence has been formulated in two slightly different ways: -Try(Ser,Ala) Val.Thr .Leu .Ala.Asp(NH,) (68, 52) or -Thr .Val.Leu.Ala .Asp(NHZ) with a second alanine residue somewhere in the chain (67). The COOH-terminal sequence of other chymotrypsinogens is also difficult, to ascertain by conventional techniques. Nevertheless, investigations concerning this sequence reveal two interesting facts: (a) The s-sulfo derivatives obtained by sulfite treatment of chymotrypsinogen and trypsinogen (52), although completely denatured, can be kept in solution in the absence of denaturing agents. Absorption spectra and amino acid analysis show that tryptophan and tyrosine are unaffected by the treatment. End-group determinations show further that no peptide bonds are being split. Thus, S-sulfo derivatives appear to lend themselves remarkably well to physicochemical investigations and structural work using enzymatic degradation (to be discussed later). (b) The availability to carboxypeptidase of the COOH-terminal sequence of chymotrypsinogen A is strictly proportional to the extent of denaturation as
150
P. DESNUELLE ANI) M. ROVERY
found by viscosity determinations. Moreover, once exposed by complete denaturation in concentrated urea, this sequence remains available when the protein is renatured by dialyzing away urea at acidic pH (67). Thus, although soluble at its isoelectric point and almost fully activatable, the renatured form of chymotrypsinogen is not identical with the native one. This point will be discussed further in the section on ribonuclease. It is likely that renaturation is made possible in some cases b y the rigid network of S-S bridges which prevents full extension of the peptide chain(s) and thus allows restoration of at least a part of the tertiary structure. In the case of chymotrypsinogen, the COOH-terminal sequence represents a kind of “frce tail” which is not, involved in a “ring” closed by S-S hridges. Such a tail may remain in an extended form even aft’erthe rest, of t8hemoleculc is renatured in a more or less strict way.
2. The Three Chains of a-Chymotrypsin (Ad) It is now known that activation of chymotrypsinogen A involves hydrolysis of a total of four peptide bonds (65). Activation itself is hrought about by the t,ryptic hydrolysis of an arginyl-isoleucine bond which forms r-chymotrypsin (A,). Then, three other chymotxypsin-catalyzed splittings occur: (a) of a leucyl-serine bond leading to d-chymot>rypsin(A2) and (b) of a tyrosyl-threonine bond and an asparaginyl-alanine bond leading either to neochymotrypsinogens or to chymotrypsins of t,he a-family (A, and A*). Since two dipeptides (serylarginine and threonylasparagine) are liberated during the process, these four splittings must have cut the single chain of the precursor into three parts. I n other terms, the final product, a-chymotrypsin (A4),must contain three open chains (Fig. 7). These chains have actually been identified by Meedom (69, 70) after performic acid oxidation, and their terminal residues are those expected from the activation scheme. a-Chymotrypsin (A4) represents an interesting example of a protein having three open chains (A, B, and C) held together by disulfide bridges. The fractionation technique originally used by Meedom involves dissolution of performic acid-oxidized chymotrypsinogen in 0.1 A; ammonia, precipitation of chain B by acetic acid, and chromatography of t’hesolut.ion on Dowex 50. Chain A is not adsorbed under these conditions. Chain C is eluted by 5 M ammonia. Pure chain A is readily obtained in this way. It is a tridecapeptide beginning with cysteic acid and ending with leucine. The NH2-terminal cysteic acid proves that chain A is the NH2-terminal sequence of the precursor. Since chain A contains only 13 residues, the activating split which converts chymotrypsinogen into the primary chymotrypsin occurs very close to the amino end, namely between the fifteenth and the sixteenth bond. This bond is actually the first bond of the sequence which fulfills the structural requirements of trypsin. A quite similar situa-
157
PROTEINS OF EXOCRINE PANCREAS
tion is encountered during trypsinogen autoactivation, when trypsin splits the sixth bond (lysyl-isoleucine bond) of the KHz-terminal sequence (71, 72).
n
TYr
I I Asp* I Ala Thr
ST
Leu
AI
Ileu Arg
Thr
I Ser
I
Asp*
I
I
I
Ala' (Asp*-
+
cy< -Neo-ChTg -
7
Ala
'
'Asp*-
T.C.
C.C.
+ Thr.Asp*
+Cys
7-
-
ChT-6
TY
€tL€52
FIG.7. Stepwise formatioil of the three chains of a-chymotrypsiri (A,) (64). ChTg, bovine chymotrypsinogen A. ChT-a, -8 and -a, respectively, r-chymotrypsin (Ad, 8-chymotrypsin (Az), and a-chymotrypsin (AJ. Neo-ChTg, neochymotrypsinogens (degraded and still activatable forms of chymotrypsinogen A). and -, NH2- and COOH-terminal residues, respectively. T.C. and C.C., hydrolysis catalyzed by trypsin and chymotrypsin, respectively. Asp*, asparagine residue. A, B, and C, chains A, B, and C of a-chymotrypsin (Al).
+
In both cases, activation is associated with a sharp decrease in optical rotation (73, 74) which suggests that the chain has become more tightly coiled. In contrast, when chymotrypsinogen A is converted into neochy-
158
P. DESNUELLE AND M. ROVERY
motrypsinogens by the splitting of a single bond in another point of the molecule, the optical rotation remains constant (75). Therefore, a certain correlation can be immediately established between activation and some rpodifications of the tertiary structure. This correlation is of course in full agreement with the accepted theories concerning the catalytic center of serine-enzymes (76). But the correlation between the precise location of the strategic bond and the structural modifications is as yet unknown. It has been postulated in the case of trypsinogen that the four negatively charged aspartic acid residues of the NH2-terminal sequence induce a local extension by electrostatic repulsion and that the chain coils further like a released spring when the residues are split off (77). Nothing indicates thus far that this hypothesis or a similar one can be applied to the chymotrypsinogen case. A second point must be raised concerning the location and general environment of the strategic bond. The main characteristic of a precursor is of course the ability to give an active enzyme after suitable modifications of its structure. But when the conversion involves, as in the present case, the splitting of a single bond, high yields of active enzyme are only obtained when the splitting of this bond is quite specific. Chymotrypsinogen A and trypsinogen contain, respectively, 19-20 basic bonds (4 arginines, 2 histidines, 13-14 lysines) and 19 basic bonds (2 arginines, 3 histidines, 14 lysines) which a t first sight fulfill the structural requirements for tryptic cleavage. Nevertheless, the strategic bond is split quickly arid quantitatively, whereas the splitting of the others, which would probably inactivate the molecule, does not occur or occurs to a very slight extent. This unique characteristic is again likely to be under the control of the tertiary structure which may expose the strategic bond and bury the others. The location of the strategic bond a t the amino end of a peptide chain is perhaps significant in this respect, although the amino end in chymotrypsinogen is not a free tail as in trypsinogen, but a ring closed by the NHP-terminal half-cystinc. To return now to chain A, Meedom (78) almost completely established the structure of this chain by the classic techniques of partial degradation. The single doubtful detail concerned the relative location of proline and alanine in the fourth and fifth positions. It is now known (67, 75) that, aminopeptidase splits cysteic acid and glycine quickly and then valine much more slowly. This fact suggests that proline may be the fourth resi-. due. When the DNP-derivative of the main peptide left behind by amino-. peptidase is submitted to partial degradation by acid, the dipeptide DNP.Val.Pro is obtained. Thus the complete sequence may be written: S
I
C y.GIy.Val.Pro.Ala.Ileu.Val.Pro.Glu.(NHa).Leu.Ser.Gly.Leu.
159
PROTEINS O F EXOCRINE PANCREAS
where Cy-S is cysteic acid in chain A and half-cystine in the NHz-terminal sequence of chymotrypsinogen A. This latter sequence may be further extended by placing on the right : Ser.Arg.Ileu.Val.Gly- which corresponds to the dipeptide split-off during activation and to the amino end of chymotrypsin. Thus, the arrangement of the first 18 residues of the NHz-terminal sequence of bovine chymotrypsinogen A is now determined. However, the situation is far from being so clear for chains B and C. These chains have been reported by Meedom to contain 180 and 50 residues respectively. But the technique described does not give pure products,
1
FIG.8. Purification of chain C by chromatography on Sephadex G-50 (79). Sephadex G-50 column (1.8 X 35 cm) equilibrated with 0.05 M HCI. Lyophilised aqueous extract of 30 mg of DFP-inhibited, performic acid-oxidized a-cbymotrypsin (A,) is dissolved in 1 ml 0.05 M HCI, put into the column, and eluted (5 ml/hr) b y 0.05 M HCl. Solid line, absorption of the fractions at 280 mp. Dotted line, absorption at 230 mp. Ordinates, optical density. Abscissas, volume of eluate in milliliters. A, chain A; C, chain C.
mainly because the chains associate strongly and are degraded in ammonia by some unknown mechanism. Better results are obtained for chain C, which corresponds to the chymotrypsinogen COOH-terminal sequence, when aqueous extracts of oxidized a-chymotrypsin (A4) are chromatographed on Sephadex G-50 equilibrated with 0.05 M HC1 (79). Figure 8 shows that three well-separated peaks emerge. The first one is an ill-defined mixture containing chains B and C. The second is chain C in an apparently pure form. The third is the small chain A which does not absorb at 280 mp. Chromatographically purified chain C can be obtained in an over-all yield of 30%. It contains alanine as the single NH2-terminal residue and all amino acids except histidine and phenylalanine. Nothing is known thus far about the further purification of chain B.
1 GO
r.
DESNUELLE AND M. ROVERY
3. Treatment with Sulftte
The specific splitting of protein disulfide bridges is an int'erestirig prohlem in several respects. (a) Cystine is partially destroyed during acid hydrolysis. An accurate determination of this amino acid requires it,s previous transformation into another derivative. (b) Intrachain and interchain bridges slow down enzymatic attack and the chromatography of tJheresult,ing cystine peptides is difficult. (e) In most proteins, disulfidt? bridges represent the only interchain link. When the bridges are cut, t,he chains tiecome independent and can be isolated. Performic acid oxidation, which converts each bridge into two sulfonic acid groups, has been successfully used in a number of cases; hut it has serious limitations. Even when special precautions are observed for minimizing chain splittings and tyrosine halogenation, destruction of tryptophan seems to be unavoidable. Besides, the oxidized material is often poorly soluble. Reduction preserves the integrity of tryptophan. All the att,empts made for split,t,ing protein disulfide bridges by metal hydrides, cyat,eine, thioglycolate, and mercaptoethanol havc heen already comprehensively reviewed. The SH groups appearing during reduction are labile and quickly reoxidize. They must therefore be converted a t once irit,o more st,ahle derivatives by alkylation. When iodoacetate or iodoacetamide is the alkylat,ing agent,, 8-carboxymethylcysteine residues are obtained which arc stable during acid hydrolysis and may be easily chromatographed. A different approach has been used recently for chymotrypsinogen A and trypsinogen. It involves treatment of the protein by sulfite in the presence of a n oxidizing agent. Sulfite (80) splits each bridge by forming an organic thiosulfate (S-sulfonic or S-sulfo compound) and a thiol. The oxidizing agent [o-iodosobeneoate, tetrathionate (81), or cupric ions (82)], transforms the thiol again into disulfide and so on until the reaction is complete. S-Sulfocysteine is unfortunately labile during acid hydrolysis. But the extlent of the reaction may be followed either hy ampernmetric titration of the thiol formed in the absence of oxidants (81), by polarographic determinatiori of t,he cuprous ions appearing when thiols are oxidized by cupric ions (52), or by radioactive techniques when S35-sulfiteis used (52). Each protein represents a new problem since certain S-S bridges are specially resistant even in denatured proteins. Under well-selected conditions, however, all bridges of chymotrypsinogen and trypsinogen appear to be split. Results obtained by cuprous ion polarography agrec with those obtained by direct determination of cystine after conversion into cysteio acid (52). Nevertheless, the possibility that the splitting is incomplete in some cases should not be neglected since it may give int,eresting information about the general structure of the molecule arid about, t,he relations between secondary structiire and activity.
PROTEINS OF EXOCRINE PANCREAS
161
Most denatured proteins are insoluble in water as a result of strong interactions between fully extended peptide chains. In the special case of S-sulfochymotrypsinogen and S-sulfotrypsinogen, aggregation may also occur. But, when special precautions are taken, water-clear solutions are obtained which are homogeneous in the ultracentrifuge (52). Under other circumstances (81), however, the S-sulfo derivatives appear to be poorly soluble in the pH range 3-9. Insolubility increases when reduction becomes more complete. No attempts have been made thus far to separate the chains of a-chymotrypsin (A4) after sulfite treatment, although this treatment is successful in the case of insulin (81). Sulfite-treated insulin is chromatographed on Dowex 50-X2 equilibrated with p H 2.2 buffer 0.2 M in citrate and 8 M in urea. Chain A passes unretarded whereas chain B is eluted by 0.2 M phosphate buffer, pH 7.6 containing the same amount of urea. Both chains have the expected compositions.
4. Primary Structure Table I gives the amino acid composition of bovine chymotrypsinogen A. Quite similar results for most amino acids have been obtained for this protein in two laboratories by the usual chromatographic technique in its manual or automatic (83) form involving all the necessary corrections and cxtrapolations to zcro time. Cystinc has been determined as aysteic acid 2Lfter pcrformic acid oxidation. Valucs of tryptophan are probably less satisfactory since they are derived from spectrophotometxic or miorobiological determinations. The protein contains only 2 histidines, 2 methionines, 4 arginines, 4 tyrosines, and 5 cystines per mole. As pointed out above, the first 18 amino acids in the NHz-terminal sequence of this protein are already known. The COOH-terminal sequence is also known, a t least provisionally, for six or seven residues. The region where chymotrypsin specifically attacks the molecule during neochymot,rypsinogen formation is -Tyr.Thr.Asp. (NH2).Ah- (65). The specific point of attachment of the diisopropylphosphoryl radical in DFP-inhibited a-chymotrypsin (A4) is G1y.Asp.Ser.Gly.Gly.Pro.Leu (89). Thus, the arrangement of 35-36 residues out of about 240 has been determined almost by chance while investigating other problems. Since bovine chymotrypsinogen A is one of the best characterized proteins available so far, it is important to launch a more general attack by conventional t,echniques. However, the enormous labor needed for the complete analysis of much smaller proteins, such as ribonuclease and lysozyme, is a good warning against excessive optimism. When digested by chymotrypsin (90-92), chain B gives an ethanol-insoluble acidic “core” with about 130 residues and 14 soluble basic peptides which seem to derive from the carboxyl end.
TABLEI Amino Acid Composition of Some Chymotrypsinogensa Bovine Ab
(84)
(85)
Bovine Bc (86) (87)
Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine
22 4 22 14 23 2 10 19 13 2 6-7 9 30 23 4 22
22 4 22 14 23 2
18 4 17 17 18 2
Tryptophan Half -cystine' Amide
7 ' 10 24
10 23
Molecular weight
25,100
-
Eikmat 280 mp N% Isoelectric point
20.0
21.0 16.5
Amino acid
16.5 9.1
10
8
19 13-14 2 6 9 27 23
15-16 10 28 6 12 17 17 3 20
4
23
-
20-21 5 19 16 20 2 8 17 10 4 6 12 18-19 20 3 21
20-21 5 18 12 19 2 9 17 10 26 5 12-13 21-22 19 4 22
6h
-
-
89
Porcine Ad (85)
8 15
79
14
21,380f 613j 24,OOOi 22,336 f 441j 22,500k 21,800 f 3%" 22,700' 18.0 18.0 16.2 16.1 5.2 7.2
NHz-Terminal residue
Half -cystine
Numbers of residues are given per 25,100gm for bovine chymotrypsinogen A. They are calculated for bovine chymotrypsinogen B and porcine chymotrypsinogen A by using the molecular weight values given b y chemical analysis (21,400or 24,000 for bovine chymotrypsinogen B ; 22,300for porcine chymotrypsinogen A). * Results obtained by ordinary chromatography for the first column and by use of an amino acid analyzer (83) for the second. Results obtained by use of an amino acid analyzer. Some of the results of the second column have been confirmed by independant methods, Results obtained by use of an amino acid analyzer. a Not included in the estimation of the molecular weight. Microbiological technique. 0 Spectrophotometric technique. Chromatography after alkaline hydrolysis. As cyst,eic acid after performic acid oxidation. j Values estimated from chemical analysis. Value obtained by sedimentation-diffusion on an impure sample (88). Values kindly determined by Mr. D. M. Brown and Prof. E. L. Smith on a sample prepared in this laboratory. The values have been obtained, respectively, b y the Archibald method of sedimentation equilibrium and by sedimentation-diffusion, assuming a partial specific volume of 0.721for the protein. J
162
PROTEINS OF EXOCRINE PANCREAS
163
A detailed discussion of the results is unnecessary since chain B has not yet been obtained in a pure form. Experiments with chymotrypsinogen are not open to the same objection. When S-sulfochymotrypsinogen is digested by trypsin (92) a core is again obtained as well as a series of soluble peptides. The core can be further digested by other proteolytic enzymes. A large number of peptides has already been identified and assembled in a preliminary way to give an unique sequence of 242 residues (93). Peptides derived from chymotrypsinogen and trypsinogen have also been extensively studied by Keil, Sorm, and their collaborators. The first experiments were mostly designed for finding out some structural similarities between both proteins. Short peptides formed by partial acid hydrolysis were isolated and compared (94-97). The usefulness of short peptides in sequential analysis is unfortunately limited. More recently, however, various larger peptides obtained by peptic degradation of chymotrypsinogen (98) and trypsin (99) and by tryptic degradation of oxidized chymotrypsin (100) have been isolated and partially identified. A list of known sequences has also been published (101).
C. Bovine Chymotrypsinogen B The second, anionic bovine chymotrypsinogen (chymotrypsinogen B) , crystallized by Laskowski et al. (46), was initially thought to be of minor importance. This impression probably arose because of heavy losses during purification. Direct chromatography (1) proved later that the amounts of chymotrypsinogens A and B in bovine pancreatic juice were in fact very similar. Therefore it became important to compare as closely as possible the structures and modes of activation of two chymotrypsinogens synthesized by the same species. End-group determinations showed some years ago that, although electrophoretically homogeneous at a single pH, crystalline chymotrypsinogen B is probably impure. It has been reported to contain chymotryptic activity and neochymotrypsinogen (87). Attempts have been made, either to crystallize the precursor in the presence of DFP (87), or to purify further the crystalline material by chromatography (102). Amino acid analysis (87) and end-group determinations (103, 104) have been carried out on these purified preparations. Another approach for purification purposes is to abandon the crystallization step. After a preliminary fractionation of 0.25 N sulfuric acid extracts of fresh pancreas, the enriched fraction is directly chromatographed (105) on a CM-cellulose column equilibrated with 0.05 M citrate buffer, pH 4.2. As shown in Fig. 9, chymotrypsinogen B is eluted by a 0.05 M citrate buffer, pH 4.6, The best fractions (specific activity, 2.5-2.6) are homogeneous as shown by an equilibrium chromatography on DEAE-cellulose in 0.05 M phosphate buffer, pH 8.0.
164
r.
DF,BNUELT,F, ANI) M. ROVERY
The amino acid composition of chymotrypsinogen B (Table I) has been recently determined in two laboratories with slightly different results. Full discussion must be postponed until more is learned about the molecular weight and exact number of residues of chymotrypsinogen B. But the general impression already emerges that both bovine precursors belong to the same protein family with low and similar amounts of arginine, histidine, methionine, phenylalanine, and tyrosine. The most striking difference is in
2c
t 3 Fractions
FIQ.9. Preparative chromatography of bovine chymotrypsinogen B (105). CMcellulose column (3.0 X 9.0 cm) equilibrated with 0.05 M citrate, pH 4.2. Arrow indicates change to 0.05 M citrate, pH 4.6. Solid line, potential activity. Dotted line, proteins. The values along the peaks indicate the specific activity of some fractions. 1. Chromatography of the precipitate obtained in 0.4 saturated ammonium sulfate (specific activity, 0.9). 11. Second chromatography of the fractions having the lowest activity in diagram I. Ordinates, activity or proteins found in each fraction (1 ml) and expressed in per cent of the total activity and total proteins introduced into the column. Abscissas, volume of eluate in milliliters. the amide content which explains a t least partly the anionic character of component B. The same holds for porcine chymotrypsiriogen A which is presented below. Bovine chymotrypsinogens A and B have the same NHz-terminal residue, namely half-cystine. Both are activated by trypsin a t about the same rate. The best technique thus far available for differentiating chymotrypsins A and B is to determine their activities on acetyl-L-tryptophan ethyl ester in the presence of 30% methanol (1). Table I1 shows that the much lower activity of chymotrypsin B on tryptophanyl esters is due to a stronger depressing effect of methanol (106).
165
PROTEINS OF EXOCRINE PANCREAS
D . Porcine Chymotrypsinogen A Comparisons between chymotrypsinogens of various species are a t least as important as between the two chymotrypsinogens of the same species. Some information is now available about porcine chymotrypsinogen and trypsinogen. Porcine pancreas is extracted as usual with 0.25 N sulfuric acid and the clear extract is fractionated by ammonium sulfate between 0.2 and 0.4 saturation. When this fraction is chromatographed on CM-cellulose, the diagram given in Fig. 10 is obtained (85). This simple technique gives a t once nearly pure products. After a second chromatography, performed under equilibrium conditions, homogeneous peaks are obtained (Fig. 11). After two chromatographic runs, porcine chymotrypsinogen A behaves as a homogeneous substance during free electrophoresis a t various pH’s and TABLEI1 Action of chymotrypsins A and B on Tyrosyl and Tryptophanyl Esters in the Presence of Methanol (106) Specific activities measured on:
ATEE”
Activated protein
A B
ATry EEa
5
30
5
30
3.0 2.2
0.81
0.92 0.70
0.78 0.05
0.60
a ATEE, acetyl-L-tyrosine ethyl ester; ATryEE, acetyl-L-tryptophan ethyl ester. The numbers below each ester represent methanol concentrations by volumee.
ionic strengths. The instability of trypsinogen prevents a closer study of its homogeneity by physicochemical means. Some of the chemical properties of porcine trypsinogen will be discussed later. The first question concerning the chymotrypsinogen is whether porcine pancreas contains one or two proteins endowed with potential chymotryptic activity. Figure 12 gives two chromatographic diagrams (85) obtained with porcine pancreatic juice collected by a permanent fistula. A potential chymotryptic activity is invariably found in the “anionic” as well as in the “cationic” fractions of the juice. When the anionic proteins obtained in both cases are further chromatographed on DEAE-cellulose a t pH 8, a large chymotryptic activity is again found in the anionic fraction. Since, although isoelectric a t p H 7.2, the chymotrypsinogen described above behaves as a cationic substance on DEAE-cellulose a t p H 8, it seems likely that porcine pancreas does contain two different chymotrypsinogens and that, the more anionic variety is inactivated, not extracted by sulfuric acid or not eluted by the phosphate gradient. For this reason, the purified precursor is named chymotrypsinogen A.
0
FIG.10. Chromatography for the purification of porcine chymotrypsinogen A and trypsinogen (85). CM-cellulose column (0.9 X 9.0 cm) equilibrated with 0.05 M citrate buffer, p H 4.3. Stepwise elution with 0.05 M buffer, p H 4.6 and pH 5.0, 10-4 M in DFP. Solid line, potential activity against ATEE (chymotrypsinogen, ChTg) and BAEE (trypsinogen, Tg). Dotted line, proteins. The values along the peaks indicat : the specific activity of some fractions. Wider columns (3.0 X 9.0 cm) are used for preparative runs, with the same results. Specific activity of the preparation put into the column, 0.9 for chymotrypsinogen and 0.19 for trypsinogen. Ordinates, activity or proteins found in each fraction (1 ml) and expressed in per cent of total activity or total proteins introduced into the column. Abscissas, volume of eluate in milliliters.
FIG.11. Second chromatography of porcine chymotrypsinogen A and trypsinogen (85). Both elutions are performed with buffers of constant composition (equilibrium chromatography). On the left : chymotrypsinogen A (specific activity, 2.9). CM-cellulose column equilibrated and eluted with 0.03 M citrate, pH 5.0. On the right: trypsinogen (specific activity, 0.350.37). CM-cellulose column equilibrated and eluted with pH G.0 buffer 0.015 M in citrate and lo-' M in DFP. Ordinates and abscissas are the same as in Figs. 9 and 10. Solid line, activity. Dotted line, proteins. 1GG
167
PROTEINS OF EXOCRINE PANCREAS
As in bovine chymotrypsinogens A and B, the single NHz-terminal residue in porcine chymotrypsinogen A is half-cystine. The amino acid composition and the molecular weight of the protein are given in Table I. .-
10
% r\
%
-
DEAE C
41%
CM-C
5
40
0
20
40
Fractions
____c
FIG.12. Cationic and anionic proteins of porcine pancreatic juice (85).On the left: DEAE-cellulose column equilibrated with 0.005 M bicarbonate, pH 8.0 and eluted after the dotted vertical line by 0.3 M bicarbonate, pH 8.3. Solid line, proteins. The figures near the peaks give the percentages of potential chymotryptic activity associated with each peak. On the right: CM-cellulose column equilibrated with 0.02 M citrate, pH 6.02 and eluted stepwise at the same pH by citrate buffers of increasing molarities (0.06,0.12, and 0.25). The figures near the peaks give again the percentages of chymotryptic activity found under each peak. Ordinates and abscissas are the same as in Figs. 9-11.
E. Bovine Trypsinogen and T r y p s i n 1. Trypsinogen Activation
An excellent correlation has been recently established, during bovine trypsinogen activation in the presence of calcium, between appearance of BAEE-splitting activity, opening of a single bond as measured by the pHstat technique, appearance of a trypsin-trypsin inhibitor peak during electrophoresis, and optical rotation changes (74). Since the same correlation is known to exist between the extent of activation, NHz-terminal isoleucine, and hexapeptide formation (72), it can now be accepted with considerable confidence that activation of the precursor is determined by some changes in the tertiary structure associated with the splitting of the sixth bond of the chain. No COOH-terminal residue has ever been found in trypsinogen and trypsin by hydrazinolysis or carboxypeptidase digestion
108
P. DESNUELLE A N D M. ROVERP
of the native or denatured proteins. Carboxypeptidase fails to liberate any amino acid even in S-sulfotrypsinogen (68). The influencc of calcium ions during t,rypsiriogen autoactivation is well known. In thc presence of calcium, activation is quantitative, in thc sensc that a given weight of precursor gives nearly the activity displayed by the same weight of crystalline trypsin. In the absence of calcium, trypsinogen gives rise to inactive proteins that are called “inert proteins” by Kunitz. The formation of these proteins has recently been investigated in some detail (107). Calcium exerts two opposite effects: (a) it speeds u p the split,ting of the strategic lysyl-isoleucine bond by a factor of more than 10 and thus great,ly accelerates the activation process. It would be int,eresting to know whether this specific influence on the sixth bond of the tail is due to a local effect, for instance on the four aspartic acid residues present nearby, or to a general modification of the tertiary structure which would expose tho bond in question still more. (0) In trypsinogen, as in many proteins, calcium protects some bonds against tryptic attack. This second effect is generally considered as brought about by a strengthening of the tertiary st8ructture which shifts the equilibrium: native form denatured form further t’otbo left. The inactive proteins formed in t,he absence of calcium have not, yct~ been fully identified, but they appear to contain additional end groups (NHZterminal serine for instance) which probably correspond to additional splits in the interior of the chain. The latter should not be considercd as merely inactivating preformed trypsin. They prevent the opening of the sixth bond by some changes in the tertiary structure of trypsinogen (107). It has also been shown by Kunitz that trypsinogen is activated b y firstorder reactions induced by other enzymes such as enterokinase and mold proteases. As in autoactivation, activation by purified enterokinase runs parallel with the appearance of an NH2-terminal isoleucine (57). But the hexapeptide has not been isolated in this experiment and enterokinasc acts in a pH range whcre autoactivation occurs a t a quick rate. More work is clearly needed before the identity of both processes is firmly established. In contrast, accurate experiments have been carried out to clear np tha mechanism of trypsinogen activation by mold proteases. These enzymes act a t acidic pH’s a t which autoactivation and further trypsin autolysis do not occur. A crystalline protease from Aspergillus saitoi fully activates bovine trypsinogen a t p H 4.5 by splitting the same lysyl-isoleucine bond as trypsin (108). Owing to the broader specificity of the mold enzyme, however, some other bonds are split in the hexapeptide as well as in the proteins formed during the process. Since activation is quantitative, this latter fact, suggests that some degraded, still active, and so far unidentified forms of trypsin may exist, Split products of the hexapeptide also appear when trypsinogen is activated by an enzyme from Penicillium janthindlum (109, 210).
PROTEINS OF EXOCRINE PANCREAS
109
2. Activation of Acetyltrypsinogen
Can enzyme molecules be simplified? If the hypothesis of a narrowly restricted catalytic center is correct, the rest of t,he molecule may he regarded as a nonessential, removable piece of “junk” (76), as long as the enzyme-substrate connections and the stability of the center are retained. Large parts of some enzyme molecules may actually be removed without impairing activity. But the existence of active split products in t,he specific case of trypsin is still doubtful. The possibility that acetyltrypsin may undergo aiitolysis without losing activity is now denied. Inactive acetylated molecules are merely digested by active ones (111). But it has been postulated recently that acetyltrypsinogen, which cannot be activated by trypsin since the t-NH2 of the sixth lysine is blocked (74), may give rise to active products under the influence of pepsin at pH 3.4 (112, 113). These products are eluted in a well-defined peak during chromatography on DEAE-cellulose. They have been reported to have a molecular weight of 6000, a single NHz-terminal residue of phenylalanine, and no histidine. This last point would shatter the accepted theories concerning esterolytic centers. For the time being, it can merely be pointed out that acetyltrypsinogen is heterogeneous (74, 113) and that the specific activity reached during peptic activation is low. But nothing of course absolutely requires that the active center of t>rypsinmust be built in a single way. 3. Chromatography
Bovine trypsinogen and trypsin are still prepared by the tedious and time-consuming technique of Northrop and Kunitz involving ammonium sulfate fractionations and crystallizations at alkaline pH. Trypsinogen can be crystallized only once and is obtained in rather impure state. Crystalline trypsin also is not pure. Besides NHz-terminal isoleucine, it contains some other end groups which are eliminated by further crystallizations of the DFP-inhibited derivative. However, bovine trypsinogen has been successfully chromatographed on Amberlite IRC-50 at pH 6.0 (1, 114) or on CM-cellulose at p H 3.2 in a gradient of increasing ionic strength (1 15). The starting materials used are commercial crystalline preparations (114, 115), acid extracts of fresh pancreas (114), or pancreatic juice (1). Results obtained with the porcine precursor (see Figs. 10 and 11) suggest that better resolution would be achieved on CM-cellulose at a somewhat higher pH. Bovine trypsin can also be chromatographed on CM-cellulose a t pH 3.2 in an ionic strength gradient (115). I n this way, active trypsin begins to separate from inactive proteins present in commercial preparations or formed during trypsinogen activation in the absence of calcium. After
170
P. DESNUELLE AND M. ROVERY
repeated chromatography, it appears to be essentially pure, but the yield is rather low and a poor scparation of trypsin and trypsinogen is obtained under these conditions. A crystalline sample of bovine trypsin has recentiy been chromatographed on CM-cellulose at pH 6.02 in the presence of calcium ions (116). The diagram of Fig. 13 show that, after the emergence of an unretarded inactive peak (25 %), trypsin can be eluted in an apparently homogeneous form by raising the molarity of the citrate buffer
I
%
20 -
0
10
0.05M
20
Fractions
4O.IOM
FIG. 13. Chromatography of active trypsin on CM-cellulose (116). CM-cellulose column (0.7 X 9.0 cm) equilibrated with pH 6.02 buffer 0.05 M in citrate and 0.013 M in CaCl2. Arrow indicates change to buffer 0.1 M in citrate and 0.013 M in CaC12. Commercial sample of crystalline trypsin (specific activity, 0.23). Solid line : proteins (25% in the first peak and 64% in the second). Activity against BAEE, 0% in the first peak and 80% in the second. Figures along the solid line give the specific activity of the fractions against BAEE. Dotted line: activity against ATEE (13y0 in the first peak, 48% in the second, and 33% in the third). Figures along the dotted line give the specific activity of the fractions against ATEE. Ordinates are the same as in Figs. 9-12. Abscissas, number of fractions having a volume of 1.4 ml.
from 0.05 to 0.1. When ATEE is used instead of BAEE for testing the activity of the fract.ions,three peaks are obtained. The first two probably represent chymotrypsin or chymotrypsin-like enzymes which are not eliminated by crystallization. The third is exactly under the trypsin peak. This latter observation suggests that pure trypsin is able to split tyrosyl esters at quite a noticeable rate. The ratio of the activities displayed by pure trypsin against ATEE and BAEE seems to be 1:6.5. On the other hand, the ratio of the ATEE-splitting activities of pure trypsin and pure chymotrypsin seems t o be about 1:50. Trypsin and chymotrypsin are extensively used for the determination of
PROTEINS OF EXOCRINE PANCREAS
171
the primary structure of proteins. Their well-known specificity enables them to split a limited number of bonds and to form relatively simple peptide mixtures. Trypsin in particular is believed to be specific essentially only for “basic” bonds and any peptides formed by this enzyme are expected to have a basic COOH-terminal residue. The total number of peptide bonds split by trypsin in a given chain is expected to be, and sometimes is (117), equal to the number of basic residues. However, trypsin as well as chymotrypsin sometimes split peptide chains at “wrong” points. These disturbing anomalies are commonly attributed to the presence in each enzyme preparation of small amounts of the other. Attempts are often made to eliminate what is considered as an undesirable impurity. But trypsin is often tested on “aromatic” substrates, such as p-nitrophenylacetate, 0-naphthyl esters of N-acylphenylalanine (1 18), fatty acid esters of m-hydroxybenzoic acid (1 19), and p-nitrophenyl esters of carbobenzoxyamino acids (120, 121). Trypsin is inhibited by aromatic derivatives of orthophosphoric acid. This suggests that cross reactivity may exist with chymotrypsin. Cross reactivity between both enzymes has actually been demonstrated by kinetic experiments in the presence of specific inhibitors (122). Figure 13 confirms this fact in the case of trypsin. About two-thirds of the chymotrypsin-like activity of the sample belong to a contamination and may therefore be eliminated, for instance by chromatography. But the rest appears to belong to trypsin itself, since the ATEE-splitting, as well as the BAEE-splitting, specific activities are constant all along the trypsin peak.
F. Porcine Trypsinogen Figures 10 and 11 show how porcine trypsinogen can be prepared in high yield by chromatography on CM-cellulose. The amino acid composition of this protein is given in Table 111, with two sets of values for the bovine precursor. Analytical results have also been obtained with a commercial sample of bovine trypsin (123). It would certainly be premature to compare the general composition of bovine and porcine trypsinogens. However, three specific differences have already been noted (126) : (a) the NHz-terminal residues of porcine trypsinogen is phenylalanine instead of valine. (b) Even in its native state, the porcine protein is attacked by carboxypeptidase A. After urea denaturation, large amounts of alanine and asparagine are liberated, followed at a slower rate by isoleucine, glutamine, threonine, and leucine. Since hydrazinolysis by the usual procedure gives negative results, asparagine is likely to be COOH-terminal in porcine trypsinogen. More experiments are needed for the rest of the sequence. (c) As in the case of bovine trypsinogen, a single significant peak is obtained by chromatography of the dialyzable compounds formed during autoactivation. This peak, however, contains the
172
P. DESNUELLE AND M. ROVERY
octapeptide l’he.l’ro.Thr.(Asp)4.Lys, instead of the hexapeptide Val(Asp),.Lys. It is quite interesting to note that the two trypsinogens available at the present time are activated by a similar mechanism, that the TABLEI11 Amino Acid Composition of Bovine and Porcine T r ypsinogensa Bovine trypsinogen Amino acid (124)
Residues (10-8) in 100 gm
Residues in 24,900 gm 15-16
-
62.5 16.5 112.1 67.7 101.2 15.3 60.4 64.9 43.1 8.1 20.9 43.5 101.2 44.3 32.2 63.3
12
-
12 35
(125)
--
Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine
-
13 2 24 10 21 3 12
2 25
11
3
-
la
14 1 4 7 38 9-1 1 9 15
14-15
-
3 8 39-40 11 4
Half -cystine Amide
23 f 3
Molecular weight E:im at 280 mp N% Isoionic point
23,800 13.9 9.3
NH2-Terminal residue
Valine
Porcine trypsinogen (126)
23,300 -
-
-
4
28 17 25 4 15 16 11 2 5 11 25 11 8 16
24,909 f 549b 13.9 16.9 Phenylalanine
a Numbers of residues are given per 23,800 gm for bovine trypsinogen. They are given per 24,900 gm for porcine trypsinogen and itlso per 100 gm, since the molecular weight of this protein calculated from chemical analysis alone is still preliminary. b Preliminary value estimated from chemical analysis.
characteristic structure (Asp),.Lys on the left of the strategic bond is the same in both proteins, and that the other, more distal residues of the NH2terminal sequence are different. In a family of proteins elaborated by various species for a given biological function, there are probably “essential”
PROTEINS OF EXOCRINE PANCREAS
173
parts, closely related with the function, which have a predetermined structure and “unessential” parts in which some variations are permissible.
G . Bovine Procarboxypeptidase A and Carboxypeptidase A 1. Purification and Activation of Procarboxypeptidase A
In 1935, Anson (127) crystallized what is now called carboxypeptidase A from autolyzed bovine pancreas and noted that fresh pancreas did not contain the active enzyme, but an inactive precursor now named procarboxypeptidase A. It has been reported in the preceding sections that pancreatic juices of other species also contain large amounts of procarboxypeptidase A which can be separated by chromatography on DEAE-cellulose at pH 8.0 in a buffer of increasing molarity. For preparative purposes, it is perhaps more convenient to start from pancreas acetone powder (pancreatin). Ninty-five per cent pure bovine procarboxypeptidase A has been obtained by ammonium sulfate fractionation of pancreatin extracts and isoelectric precipitations (47). When the proteins precipitated by 0.39 saturated ammonium sulfate are chromatographed on DEAE-cellulose in a concentration gradient of pH 8.0 phosphate buffer, the two last and most acidic peaks contain procarboxypeptidase A in an electrophoretically homogeneous form (48). The molecular weight of the protein determined by light scattering and sedimentationdiffusion is 94-96,000. Its isoelectric point in univalent buffers of 0.2 ionic strength is below 4.5. The tryptic activation of bovine procarboxypeptidase A into carboxypeptidase A has already been discussed in a series of comprehensive reviews (7, 128). It will be recalled here that in sharp contrast to trypsin and chymotrypsin, carboxypeptidase A is formed by only one-third of the precursor molecule, the rest being split off during activation. But the most interesting point of the activation process is probably the transitory appearance of a ATEE-splitting, DFP-sensitive enzyme which accumulates at low temperature in the presence of low amounts of trypsin (48). When the temperature and the amount of trypsin are raised, a part of this chymotrypsin-like enzyme disappears, whereas carboxypeptidase activity, measured against carbobenzoxyglycylphenylalanine,appears. It had been assumed that procarboxypeptidase A gave rise in a first step to the ATEEsplitting enzyme, which was converted later on into carboxypeptidase A by autolysis and further degradation by trypsin. However, it has been shown more recently (129) that procarboxypeptidase A is split by concentrated urea, buffer solutions at pH 10, and chelating agents such as 1 ,lophenanthroline into three subunits with the same sedimentation rate as carboxypeptidase A. It is also now known that procarboxypeptidase A
174
P. DESNUELLE AND M. ROVERY
contains three NHz-terminal residues, aspartic acid (or asparagine) , lysine, and half-cystine. Therefore, the possibility exists that the ATEE-splitting enzyme and carboxypeptidase A do not arise, as believed earlier, successively from the same molecule, but from two different subunits of this molecule. Bovine carboxypeptidase A is likely to be formed by a single chain beginning with an asparagine residue (130) and terminated by the sequence (Glu,Leu,Thr,Val) Asp (NH2) ( 131). 2 . Role of Metals in Carboxypeptidase A
Unlike chymotrypsinogen and trypsinogen, carboxypeptidase A is not inhibited by DFP and cannot therefore be considered as a serine enzymp. It is actually a metallo enzyme containing one zinc atom per molecule.2 When zinc is removed by dialysis against acidic buffers or 1,10-phenanthroline (132, 133), an inactive apoenzyme is obtained which does not differ from the enzyme itself, except for activity and zinc content. Activity is gradually restored by addition of zinc up to one equivalent (133, 134). Some other metals of the first transition period (Mn++, Fe++, Co++, Ni++) can also restore activity in the apoprotein (134). Relative affinities of the apoenzyme for various metal anions have been investigated by equilibrium dialysis against solutions of radioactive salts (134). It is now demonstrated that zinc is bound in the bovine enzyme to an SH group probably belonging to a cysteine residue (135). I n contrast to the native or reconstituted carboxypeptidase, the apoenzyme has one titratable SH group. A good correlation is found between titratable SH group, zinc content, and activity. Long storage induces simultaneous loss of SH groups and loss of reactivability by zinc. Both are partially restored by a limited addition of cysteine. Furthermore, apocarboxypeptidase A treated by zinc liberates two protons, indicating zinc binding to a second group thought to be nitrogen (136). Thus, the active center of the enzyme is likely to include one sulfur, one nitrogen, and one zinc atom. Some observations also suggest that the nature of the metal bound to the apoprotein may influence the activity of the enzyme against different substrates. Cadmium, mercury, and lead carboxypeptidases have been reported to be active on esters only (136). On the other hand, when the zinc-containing, active carboxypeptidase is incubated with cobaltous ions, activity towards the specific peptide substrates increases two-fold whereas esterase activity to wards hippuryl-L-phenyllactic acid remains unchanged (137). While acting in this interesting way, cobaltous ions are much less tightly bound to the protein than zinc ions. Original activity is quickly regained by dilution or dialysis. A bibliography of this subject is found in a recent and comprehensive review by Neurath (128).
PROTEINS O F EXOCRINE PANCREAS
175
H . Bovine and Porcine Carboxypeptidases B Certain bonds are split by proteolytic enzymes, not merely because they belong to a given protein, but because their structure, locatJion, and/or availability fulfill certain requirements. Thus, the nomenclature of proteolytic enzymes should not be based upon the name of the proteins of which these enzymes have been tested for the first time, but upon factors directly connected with the bonds. A good illustration of this important principle is given by the following case: It was claimed that pancreas contains a “protaminase” because autolyzates of the gland displayed considerable activity against protamines (138, 139). It turned out later that pancreas actually contains a second carboxypeptidase which easily splits the basic bonds of these peculiar proteins. The specific range of this second carboxypeptidase has been unambigously defined (49-51). It hydrolyzes COOH-terminal basic bonds in synthetic substrates such as a-N-benzoylglycyl-L-lysine and hippuryl-Larginine as well as in proteins. In contrast to carboxypeptidase A, it is competitively inhibited by eaminocaproate and 6-aminovalerate (50). Thus pancreas contains a basic carboxypeptidase (carboxypeptidase B) which exclusively splits off the COOH-terminal basic residues appearing during tryptic hydrolysis, and a carboxypeptidase A which preferentially splits off the COOH-terminal aromatic residues resulting from previous chymotryptic digestion. Like carboxypeptidase A, carboxypeptidase B exists in pancreas and pancreatic juice in the form of an inactive precursor, procarboxypeptidase B. Bovine procarboxypeptidase B has been purified from pancreatin extracts (50). However, after a twenty-fold purification according to the technique described, the preparation still contains large amounts of chymotrypsinogen B ( 5 2 ) .Final purification must therefore involve chromatography as well as fractional precipitations and extractions. Porcine procarboxypeptidase B presented in the chromatographic diagram of Fig. 2 has not yet been further characterized, though an apparently good purification technique is now available for porcine carboxypeptidase B (53). This technique includes water extraction of an acetone powder of autolyzed pancreas, fractionation with ammonium sulfate, and chromatography on DEAE-cellulose columns eluted with a linear gradient of 0.0 to 0.2 M NaCl in 0.005 M Tris buffer, pH 7.5. It is quite significant that all procedures recently described for the purification of pancreatic proteases are based upon some preliminary fractionation with ammonium sulfate and a highly efficient ion-exchange chromatography under suitable conditions. When purified in this way, porcine carboxypeptidase B is electrophoretically homogeneous in a 0.05 2cI phosphate buffer, pH 7.02. Sedimentation-
176
P. DESNUELLE AND M. IE0VEH.Y
diffusion gives for its molecular weight the same value as for bovine carboxypeptidase A, namely about 34,000. It also contains one zinc atom per mole (53) which can be removed and replaced by other metal ions. I n the case of carboxypeptidaseB also, the natureof the metal bound to the enzyme appears to influence the ratio peptidase activity: esterase activity. Incubation with cobalt enhances peptidsse activity and incubation with cadmium TABLE IV Aniino A c i d CoinpositiorL of Bovine liarboxypeptidase A and Porcine Carboxypeplidase B Number of residues per 34,400 gm of: Amino acid
Bovine carboxypeptidase A (141)
Alanine Arginine Aspartic acid Cystine (half) Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methioiiine Phenylalttniiie Proline Rerine Threonine Tryptophan Tyrosine Valine
20.0 10.0 30.3 4.0 25.0 23.2 7.7 20.1 24.7 18.4 1.0 14.9 11 .0 33.1 26.6 6.1 19.7 16.4
25.1 10.0 32.4 7.6 24.8 23.0 5.8 17.2 22.7 17.5 5.1 11 .n 13.2 17.5 30.2 9.2 20.4 10.8
312.2
304.4
-
Porcine carboxypeptidase B (53)
cnhzlnaes csterasc activity (140). Table I V gives the amino acid composition of bovine carboxypeptidase A and porcine carboxypeptidase B.
I . Porcine Lipase The purification and properties of porcine pancreatic lipase have been discussed in a recent review (142). Only the most significant facts related to the protein nature of the enzyme will therefore be presented here. 1 . Purification
After many unsuccessful attempts, porcine lipase has been recently obtained in a satisfactory state of purity. Aqueous extracts of porcine pancre-
177
PROTEINS OF EXOCRINE PANCREAS
atin are extracted with water. The extracts are fractionated by ammonium sulfate and acetone (143) as well as by selective absorption on tricalcium phosphate and aluminium hydroxide (16). The last step of the purification procedure is a zone electrophoresis on starch in a high potential gradient (16, 143). As shown in Fig. 14, an electrophoretically homogeneous protein
3530)
I
40
rnl
FIG.14. Electrophoretic homogeneity of porcine lipase (18). Starch columns (3.0 X 90 cm) equilibrated with 0.025 M acetate buffer, p H 5.25 (diagram on the left), or 0.025 M veronal buffer, pH 8.0 (diagram on the right). Potential gradient inside the column, 8 volts/cm; temperature, +l"C.Elution after 48 hr. Ordinates, per cent in 1ml eluate of the total lipase activity (solid line) and total proteins (dotted line) introduced into the column. The vertical dotted line gives the true origin, with due regard t o the electroosmotic flow. The figures along the peaks give the specific activity of some fractions. Specific activity of the sample introduced into the column, 3500. Abscissas, volume of eluate in milliliters.
is finally obtained after a 135-fold purification with a 20% yield (16). This protein is also homogeneous during chromatography on tricalcium phosphate. An additional proof of purity is given by the fact that the same maximal specific activity is reached, but not surpassed, when purification starts from porcine pancreatic juice (17). The diagram of Fig. 2 suggests that lipase could also be purified by chromatography on DEAF,-cellulose at pH 8.0. A series of other chromatographic techniques are not, siiitable for preparative purposes (144).
178
P. DESNUELLE AND M. ROVERY
Porcine pancreas and porcine pancreatic juice appear to contain a single protein endowed with lipolytic activity. As stated earlier, this protein corresponds to about 2.5 % of the total proteins of the juice. Its molecular activity (turnover number) is likely to be higher than 300,000 under the conditions of the test. Shortage of pure material has thus far prevented any investigation of its molecular properties. It is merely known to be quite soluble in water, to have an isoelectric point of 5.2 in 0.025 M acetate buffer, and to give a conventional protein spectrum. Lipase present in pancreatic juice is likely to be identical with the enzyme extracted from pancreatin. 2. Interactions with Insoluble Substrates and Inhibitors Porcine lipase exhibits a series of unusual properties (142). The most interesting one, directly connected with its structure, is certainly its specific interactions with emulsified esters. When an aqueous solution of lipase at pH 5 is mixed with an excess of a triolein emulsion and when the cream is separated by centrifugation, no lipase can be detected in the clear aqueous lower phase. All activity is in the cream. But it returns at once into water when the emulsion is broken (145). This simple experiment suggests that lipase is adsorbed at the oil/water interface of the emulsion and that this interface may be the normal site of its action. Such an assumption is confirmed by a series of experiments with substrates and inhibitors. a. Substrates. In the usual case of a water-soluble enzyme acting upon a water-soluble substrate in an homogeneous or “microheterogeneous” phase, a fully reversible equilibrium is assumed to exist between enzyme, substrate, and some kind of primary enzyme-substrate complex. This complex is further degraded at a given rate either directly or after an intramolecular “activation.” When substrate concentration is raised, the initial velocity of the reactJionincreases up to a point a t which all or almost all enzyme molecules are converted into the complex. Figure 15 shows that this classic picture does not hold for lipase. Pure lipase does not hydrolyze methyl butyrate and triacetin as long as the esters are dissolved in water. Conversely, lipolysis starts as soon as, by oversaturating the solution, some ester molecules begin to appear in an emulsified state. Thus, pure lipase hydrolyzes ester emulsions, not ester solutions (146). The same fact is demonstrated in a different way by the electrophoretic diagram of Fig. 16. When fractions isolated by zone electrophoresis of an impure lipase preparation are tested on ester emulsions, all the activities are found under a first peak which is the lipase peak. When the same fractions are tested on ester solutions, activities are found under another, wellseparated and more anionic peak (146, 147). The final proof that lipase is acting at the interface itself, and not inside the emulsified globules, is given by the following experiment: A coarser and
I
/+T-
0'1 O 2 0.30.40.5 O 6 d.7 08,i I6
Soluble
nsoluble I
5-
4-
20
3-
2I-
0 5s
1.0s (0.328M)1.5s
2 0s
/I
3 S
2 0 o
0.5s L 0.5s m
,-. A, , c JI.OS(0.153~) 1.5s , ' I.OS(0.153~) 1.5s
,
2,.0s 2.0s
,
I
FIG.15. Exclusive action of porcine lipase on emulsified esters (146). Ordinates: activity in per: cent of maximal activity on triolein emulsion. Abscissaa: lower axis, substrate amounts expressed in fractions of saturation for the solutions (on the left of the vertical dotted line) or in multiples of saturation for the emulsions (on the right of the line); upper axis, interfacial area expressed in lo6X cme in 100 ml. White circles, impure lipase containing some esterases. Black triangles, purified lipase. The substrate is triacetin on the left and methylbutyrate on the right.
180
1’. DESNUELLE AND M. ROVERY
a finer triolein emulsions are prepared. Initial velocities of the reaction induced by the same amount of lipase under the same conditions are plotted in each case against substrate “concentration.” When this concentration is expressed by the weight of the insoluble substrate in a given volume, two widely different curves are obtained. The velocity is much lower, for a
FIG. 16 Electrophoretic separation of the lipase and esterase activities of porcine pancreas (146, 147). Starch columns equilibrated with 0.025 M acetate buffer, p H 5.25. The activities of the fractions have been determined: ( a ) on emulsions of triolein and tributyriri (black circles), methyl oleate, methyl Iaurate, and p-nitrophenyllaurate (black triangles). ( b ) On solutions of methyl butyrate and p-nitrophenylacetate (crosses). White circles and dotted line, protein background. Figures along the first peak give the specific activity (lipase) of some fractions, determined against triolein emulsion. Ordinates and abscissas are the same as in Fig. 14.
givrii Wright, with the coarser emulsion than with the finer one. When
concentration is expressed by the interfacial area in a given volume (“interfacial concentration”), the curves obtained with both emulsions are almost identical. Furthermore, when different emulsions are prepared with the same weight of txiolein, the initial velocity of lipolysis depends upon the interfacial area of the emulsions. I n other words, a Michaelis curve of the ordinary shape can be drawn with lipase for a constant weight of insoluble substrate. It has been definitely established that this curve describes the gradual adsorption of lipase by an interface of increasing area. Then, a
PROTEINS O F EXOCRINE PANCREAS
181
Michaelis contant can be defined for lipase as being that interface which gives to the lipolytic reaction the half of its maximal velocity. This constant is independent of the amount of lipase used (148). b. Organophosphate Inhibitors. Porcine lipase is known to be unaffected by DFP and diethyl-p-nitrophenyl phosphate which inhibit most esterases by combining specifically with their active serine. I n fact, lipase is unaffected by organophosphate solutions. It is quickly and irreversibly inhibited by diethyl-p-nitrophenyl phosphate emulsions in a first-order reaction (149). To sum up, porcine lipase seems to be a very unusual protein which exhibits esterolytic activity when adsorbed a t an oil/water interface. Since no colipase has ever been discovered and since the enzyme itself can be inhibited by organophosphates, it may be postulated as a working hypothesis that lipase contains an esterolytic center built according to the same rules as are the centers of other esterases. The fact that lipase acts exclusively on emulsified esters and is exclusively inhibited by emulsified diethyl-p-nitrophenyl phosphate suggests further that the actual formation of this center is a consequence of interfacial adsorption. I n other words, lipase in solution, like chymotrypsinogen and trypsinogen, would he an inactive protein capable of conversion into an active enzyme by some limited changes in its tertiary structure. Instead of being induced by the splitting of a covalent bond, these changes would result in the case of lipase from interfacial adsorption (149).
J . Bovine Ribonuclease Bovine ribonuclease (150, 151) is a stable protein of relatively low molecular weight which contains no tryptophan. Extensive investigations have been carried out in recent years on its general structure and the origin of its enzymatic activity. However, the question of how many ribonucleases exist in the pancreas of beef and other species is still unsettled. When the crystalline enzyme or a sulfuric acid extract of bovine pancreas is submitted to partition (152) or ion-exchange chromatography on Amberlite IRC-50 (153), a major (ribonuclease A) and a minor (ribonuclease B) active peak are obtained. When phosphate or sucrose extracts of bovine pancreas are chromatographed on Amberlite IRC-50, three active peaks appear (154). Two of them are probably formed by ribonucleases A and B. Acid treatment of the third one converts it into the others and increases activity. Finally, chromatography of crystalline bovine ribonuclease on CM-cellulose in phosphate (155) as well as in univalent (156) buffers yields four active peaks. Recent investigations have been mostly performed on chromatographically homogeneous ribonuclease A.
182
P. DESNUELLE AND M. ROVERY
1. Amino Acid Arrangement and Location of the Disulfide Bridges
Amino acid arrangement and location of disulfide bridges in bovine ribonuclease have been completely elucidated in the last few years (157) by using a number of experimental approaches similar to those described by Sanger 10 years ago and employed by him for the determination of the covalent structure of insulin. This achievement is especially noteworthy since the single chain of ribonuclease contains 124 residues, that is to say, more than four times as much as the longer chain of insulin. Highly refined techniques were therefore required among which chromatography on cation-exchanger columns, instead of paper chromatography and electrophoresis, played a major role. Peptides were separated by manual chromatography and their amino acid composition was established by the use of an automatic amino acid analyzer. In a first series of experiments (158), performic acid-oxidized ribonuclease was digested by trypsin, chymotrypsin, or pepsin. The peptides arising during tryptic hydrolysis were taken as building stones of the whole sequence. Some of them could not be fully analyzed a t once, but all could be assembled together in the right order by taking advantage of “overlapping” sequences found in chymotryptic and peptic hydrolyzates. Then, the structures of the larger tryptic peptides were elucidated (159) and the complete sequence (160) was finally written as indicated in Fig. 17. It must be clearly realized that each shorter or larger peptide had to be obtained in pure form by chromatography and then analyzed. For the larger peptides, analysis required further breakdown and chromatographic fractionations. For the smaller ones, it required full elucidation of the structures by end-group methods and stepwise degradation by carboxypeptidase, aminopeptidase, and thiohydantoin cyclization. But the most striking point in the work is perhaps that yields were always determined, in order to see whether the sequence under investigation belonged to a single molecular entity, to a possible impurity, or to molecules derived from the principal components by some biosynthetic fluctuations around an average model. Yields were sometimes excellent. They could always be considered as satisfactory when unavoidable experimental losses were taken into account. All peptides were fully analyzed and found t o fit into the unique sequence of Fig. 17. To the best of our knowledge, therefore, the ribonuclease used appears to be a chemically homogeneous protein preparation, that is to say, a preparation in which all the molecules have the same covalent structure. This important observation suggests that the residue arrangement may be strictly determined even along a relatively large peptide chain and that, a t least in the case considered here, no fluctuation leading to a somewhat confusing microheterogeneity is taking place during biosynthesis of a given protein. Moreover, ribonuclease is the first enzyme for which full structural
F
-
-
rNH2
-.
Ala-Ala~Lys-Phe-Glu-ArqSer -Thr 5er-5er -Asp -His-Met-Glu-Ala-Ala
Thr
m
11 FIG.17. Amino acid arrangement and disulfide bridges in bovine ribonuclease (157). The arrows indicate the direction of the chain starting from the amino end. The heavy black links represent disulfide bridges.
184
P. DESNUELLE AND M. ROVEItY
information is available at the covalent level. The case of an enzyme is especially interesting since, as already stated, enzymatic activity is likely to be associated with the existence of a catalytic center. The results dewribed above prove that not only the nucleolytic center but also the entire ribonuclease molecule may have a predetermined structure. This does not mean of course that chemically different molecules cannot have the same activity, but that the residue arrangement may be strictly controlled well outside thc enter.^ Some years ago, Sanger also developed an elegant technique for locating the disulfide bridges of a protein. It includes partial degradation of the intact molecule, separation of the cystine peptides, and identification of the two cysteic acid peptides arising during performic acid oxidation of each cystine peptide. However, when insulin was submitted to degradation by enzymes and acid, more cystine peptides were formed than could be expected from the number of cystine residues. The reason for this anomaly is that disulfide interchanges are taking place during the degradation step leading to cystirie peptides. At least two types of reactions are involved, one inhibited by thiols in strong acid and the other catalyzed by the same thiols in neutral or alkaline solutions (161). In the last case, interchange is likely to be initiated by the well-known hydrolytic fission of disulfides. RiSSRl
+ OH- 2 RiS- + RISOH
As so011 as R& ailions are formed, the propagatioii of thc rcactioi: is induced hy tho following cyuilihris: 1tzSSlL II3SSRa
+ It&
+ It&
;2
RlSSjHL
2 RzSSR3
+ R,S-
+ It&,
etc.
At neutral or alkaline pH’s, interchanges are slowed down when the RSform is blocked by sulfhydryl reagents such as N-ethylmaleinimide or p chloromercuribenzoate. All disulfide bridges of insulin have been correctly located in this way (162). Unfortunately, the reaction of the RS- form drives the first equilibrium low-ard the right and consequently decreases the amount of the primitive disulfide bridges. Some bridges appear to be quite labile under these circumstances and their recovery is very low. This may lead to serious mistakes. Rovinr ribonurlease contains eight half-cystine residues which are Recent investigations at the National Hetlrt Institute, Bethesdtl, Maryland, and the Rockefeller Institute for Medical Research, New York, indicate that the sequence of ribonuclease involving residues 11 through 18 is incorrect as shown in Fig. 17. The correct sequence will be published shortly by both of the laboratories (personal communication from C. B. Anfinsen).
PHOTEINS OF EXOCRINE PANCREAS
185
numbered from I to VIII starting from the amino end of the chain. Two bridges (I-VI and IV-V) can be determined after degradation by subtilisin (163). The other two (II-VII) and III-VIII) are apparently destroyed or isomerized under these conditions. But they are found in 2 0 4 7 % yield after degradation by pepsin a t pH 2 followed by a short hydrolysis with trypsin and chymotrypsin (157). Pepsin seems to be the enzyme of choice for starting the degradation, since it is active a t slightly acidic pH’s, its specificity is broad, and it attacks proteins without requiring the help of a denaturing agent which may further labilize the bridges. A three-dimensional model of the ribonuclease molecule would be more significant than the two-dimensional representatiqn of Fig. 17. However, it may be noted that the ribonuclease structure is tightly coiled. Five parts may be roughly distinguished in the chain: two tails at both ends (NHZterminal tail, 25 residues; COOH-terminal tail, 14 residues) which are free of any S-S bridges, and three rings closed by S-S bridges. Eight residues are found in the smaller ring, as compared with six in the case of insulin, vasopressin, and oxytocin. The size of the larger rings (28 and 34 residues, respectively) is of the same order as in insulin (27 residues). 2. Correlation between Structure and Activity a. The Tertiary Structure. Complete elucidation of the covalcnt structure of an active protein may appear as not very rewarding. No correlation can hr established immediately brtween structure and activity. This fact is not
srirprisirig in the casr of insulin and other hormones since the exact mode of action of this class of substances is still unknown. But catalysis induced by enzymes can sometimes be expressed in molecular terms and it is probably displayed by a restricted part of the molecule. Hence, the situation is more hopeful for enzymes than for any other active proteins thus far investigated. Enzymatic activity, however, is not merely associated with covalent structures, but chiefly with tertiary structure which is still more difficult to determine. The crucial role of tertiary structure is proved by the fact that denaturation brings about inactivation. Even with proteins which may be reversibly denatured, such as chymotrypsin and trypsin, activity is lost as long as denaturation persists. Ribonuclease appeared for a while to be an exception, since it was still active in 8 M urea. But it was shown later that phosphate ions, a t a concentration as low as 0.003 M , and polyphosphates induced in urea-denatured ribonuclease spectral changes usually associated with refolding (164). It could then be assumed that ribonucleic acid, the actual substrate, was also able to refold the denatured form and prevent inactivation in this way. In other words, even in ribonuclease, the active center is probably not built by adjacent residues in a tail or a ring, but by some residues correctly located in space by the superimposed
186
P. DESNUELLE AND M. ROVERY
tertiary structure. About 15-41 % of the chain is present in an a-helical configuration in the native enzyme (165, 166). Most of this configuration is lost when the protein is denatured. Precise studies have been carried out in order to correlate ribonuclease inactivation and denaturation. The rate of ribonucleic acid hydrolysis normally increases with temperature up to 50-55°C. Its slower increase and later its decrease at higher temperatures suggest that the enzyme is inactivated afterward (167). Temperatures of 50-55°C correspond exactly t o the range in which, as shown by intrinsic viscosity and optical rotation determinations, the protein starts to unfold (168). All the observations concerning the opposite phenomenon, that is to say, the activation of an inactive precursor (see above), are also consistent with the view that active centers are dependent on tertiary structure. It must be recognized that the role played by tertiary structure complicates to a great extent the already heavy task of protein chemists. However, the full elucidation of the covalent structure of an enzyme is of fundamental importance for many reasons. It represents the first and compulsory step toward a better understanding of the tertiary structure. It allows also a closer chemical approach to the problem of enzyme inactivation and activation. The only condition is to keep in mind that, however specific and local at first sight, any covalent change may exert long-range actions on the tertiary structure. Several examples are found in the older literature of enzymes which are inactivated as soon as some of their “unreactive” groups begin to be involved in a chemical reaction. When unreactive groups are forced to react, the tertiary structure of the protein is likely to be modified and the inactivation may be attributed to this modification (169). b. The NH2-Terminal Sequence. When bovine ribonuclease A is incubated with crystalline subtilisin a t 3°C and p H 8.0, a single bond is split without impairing activity (170-173). Chromatography of the digestion mixture shows the presence of about 10% of the original enzyme and 70% of another active protein (RNase-S) moving more slowly on Amberlite IRC50. This latter is certainly different from ribonuclease since it contains an additional NHz-terminal serine residue, is quickly inactivated by trypsin, and does not display the characteristic activity in 8 M urea. But it has the same specific activity in water against a series of specific substrates, the same amino acid composition, and the same heat stability. It cannot be fractionated by dialysis against water, chromatography, and ammonium sulfate fractionation. Thus, the simplest idea is to suppose that a peptide bond has been split in a ring and that ribonuclease has been converted into a new active protein with two open chains bound by S-S bridges. However, when the digestion mixture is precipitated by trichloroacetic acid or is dialyzed against 8 M urea, a protein (S-protein) and a peptide (S-peptide) are separated. The peptide contains 20 residues which cor-
PROTEINS OF EXOCRINE PANCREAS
187
respond exactly to the first 20 residues of the NHz-terminal sequence of ribonuclease. Both parts are inactive when taken separately. But their mixture in equimolar amounts (RNase-S’) is almost as active as the original enzyme. It appears therefore that subtilisin splits in quite a specific way the twentieth bond linking an alanine and a serine residue in the KHzterminal tail of ribonuclease and that the liberated peptide remains tightly bound to the rest of the molecule. This latter, containing 104 residues, is not active by itself, but it is activated by its close association with the peptide. Although no rational interpretation can be presented for the time being, these results deserve the utmost attention. Why does subtilisin, known for its broad specificity, split a single bond in ribonuclease under a given set of conditions? Why does this splitting actually inactivate the molecule? Why does trypsin become able to attack the chain after this primary splitting? Why is the S-peptide so tightly bound to the S-protein in a way reminiscent of the association between neurophysin and the two hormonal peptides vasopressin and oxytocin (174)? How finally can inactive S-protein containing 104 residues become an active ribonuclease through its association with the S-peptide? An experimental approach to the last two problems is to modify the S-peptide groups and to determine the influence of these modifications on the strength and activity of the association. Several derivatives of S-peptide (la-hydroxy ; lc-, 7ediguanidino; la-, 16-, 7etriguanidino; la-acetamido-1 e, 7e-diguanidino; la-, le-, 7e-triacetamido; tetramethyl ester; sulfonium or sulfone derivatives of methionine on position 17) have been prepared. Their ability to associate with S-protein and to form with it an active enzyme have been investigated (175-177). Two possible explanations of the activating effect of the peptide may be considered. First, the peptide may contain a part of the active center and the association of peptide-protein may be so intimate that the whole organization of the center is retained even after the splitting of the twentieth bond. Second, all parts of the center may be present in the rest of the protein and its configuration, by itself labile, may be stabilized or even reformed by the peptide. c. The COOH-Terminal Sequence. Ribonuclease is very quickly inactivated by pepsin. Inactivation runs parallel with the appearance of the COOH-terminal tetrapeptide Asp.Ala.Ser.Va1 (178, 179). Pepsin action is probably more complex than the specific splitting induced by subtilisin. Nevertheless, an inactive preparation with the same sedimentation constant as ribonuclease and having no additional NHz-terminal residue can be obtained in high yield by chromatography of the peptic digest. It seems therefore quite possible that one or several of the four last residues of the chain have something to do with activity. d. Speci$ic Reactions with Iodo- or Bromoacetate. Ribonuclease is easily
188
P. DESNUELLE AND M. ROVERY
inactivated by iodoacetate (180) and bromoacetate (181).The first interest of this observation is to show that reagents thought to be specific for protein SH groups and even used sometimes for defining the sulfhydryl character of some enzymes may act on and inactivate enzymes devoid of SH groups. Many investigations carried out on the so-called sulfhydryl enzymes with inappropriate techniques and unspecific reagents are of little significance. The very unusual pH-dependence of ribonuclease inactivation by iodoacetate led to a careful study of the groups involved at various pH’s (180). The technique was to identify the carboxymethylated residues by chromatography after hydrolysis of the modified protein. It was shown in this way that lysine is mostly involved a t higher pH’s and methionine a t acidic pH’s. After reaction at pH 5.5-6.0, a chromatographically homogeneous and inactive protein was isolated which differed from riboriucdease only by carboxymethylation of a single histidine residue (180). By using C14-labeled bromoacetate, it was confirmed that total inactivation was brought about by alkylation of a single histidine residue arid it was shown that this residue is the one hundred and ninteerith one in the peptide chain (182). This beautiful correlation between alkylation of a single group and inactivation can be compared with the one existing between phosphorylation of one serine residue and inactivation of DFP-sensitive esterases. It raises the question of the unusually high reactivity of the group concerned and the role played by this group in the catalytic center. The crucial importance of the tridimensional environment on the reactivity of protein groups is again stressed by the fact that the histidine residue is alkylated a t a normal rate in denatured ribonuclease and that, in contrast, the reactivity of the methionine residues is enhanced by denaturation (183). A similar situation is met with subtilisin-modified ribonuclease. In the inactive, presumably denatured S-protein methionine residues are easily alkylated a t pH 6.0 by iodoacetate and the resulting derivative can no longer bind the S-peptide. But, alkylation of the presumably native association Sprotein S-peptide yields large amounts of carboxymethylhistidine (184). I n other words, the time-honored concept according to which denaturation increases group reactivity by unfolding the chain(s) is valid for “ordinary” groups. There exists some groups involved in the active center aiid some bonds involved in the activation of precursors to which a special coiling or folding of the native chain gives a very high reactivity. This reactivity actually decreases when the native configuration is lost. It is also interesting to note that the one hundred and ninteenth histidine residue and the Phe-Asp bond split by pepsin (see above) are located very close to each other in the COOH-terminal tail of ribonuclease. Inactivation occurs readily when this part of the molecule is modified.
+
PROTEINS O F MXOCHINE PANCREAS
189
e. Reduction and Reoxidation. It has been known for several years that the four disulfide bridges of ribonuclease are split by reductive agents to give an inactive product and that the extent of inactivation roughly parallels the extent of reduction as measured by the number of SH groups. Thioglycolic acid has now been shown to contain polythioglycolides formed by polymerization of thioesters. These polymers are able to react with the protein amino groups (thiolation) and thus to alter significantly the results due to reduction alone. Freshly distilled thioglycolic acid or mercaptoethanol must therefore be used (185). New technical refinements have been described for the full reduction of ribonuclease by mercaptoethanol (186). They include treatment of the protein at p H 8.6 in 8 M urea and elimination of the low molecular weight compounds by passage through Sephadex G-25 equilibrated with 0.1 M acetic acid. The SH groups are fairly stable in dilute acetic acid solution. They may be s t a b b e d further by specific alkylation under well-selected conditions or reaction with p-chloromercuribenzoate. In this latter case, SH groups may be liberated again at will and the reduced protein again obtained in a pure state by mercaptoethanol treatment and chromatography on Sephadex. It had already been pointed out 2 years ago that reduced ribonuclease regains some activity when exposed to air at p H 8.0 (187). An 80-100% reactivation has now been achieved by avoiding thiolation and surface denaturation by air bubbling. (185, 186). This reactivation is very interesting since random combination of eight SH groups to form four disulfide bridges may be expected to give 105 isomers (188). Thus, the question arises whether all the possible isomers of reoxidized ribonuclease have the same activity, or whether the original isomeric form is almost entirely regained during oxidation. The latter assumption seems to be substantiated by a comparative study of native and reoxidized ribonuclease. No difference can be detected in the chromatographic behavior of both substances, fingerprinting of cystine peptides formed by subtilisin degradation, isoionic point, optical rotation, intrinsic viscosity, immunological properties (189), and geometry of the molecules as revealed by X-ray diffraction (190). Some minor changes, however, are noted between the ultraviolet spectra of' both proteins and between their ability to give crystals of high quality. Final proof of the identity of disulfide bridges in native and reoxidized ribonuclease will probably be given by a closer study of the cystine peptides arising during pept]ic degradation (157) of both proteins. The results already obtained suggest that the primary structure, that is to say, the amino acid arrangement along the ribonuclease chain, fully determines a unique mode of attachment of half-cystine residues, as well as a unique helical coiling and folding of the chain. The necessary information for the
190
P. DESNUELLE AND M. ROVERY
determination of these structures seems to be contained in the first 104 residues] since reoxidized S-protein is activated by S-peptide (191).
K . Sheep and Mouse Ribonucleases As already pointed out earlier, it is important to purify the same enzyme or the same precursor from several species in order to compare the general structure of the molecules, the structures of their active centers, or the mode of activation of the precursors. Chromatography of sulfuric acid extracts of sheep pancreas on CM-cellulose equilibrated with 0.01 M phosphate buffer, pH 6.0 gives an initial active peak near the solvent front and then four other peaks when a concentration and pH gradient is applied to the column (155). These four peaks may be further purified under the same conditions and they behave in exactly the same way during repeated chromatography. Therefore they probably represent four different sheep ribonucleases which have been numbered I, 11, 111, and IV. The behavior of the first peak is more complex. When passed through a DEAE-column, it gives rise to one major, one minor, and two very small peaks. But, after passage through Amberlite IRC-50, very little active material is found at the original position on CM-cellulose. Most of the activity migrates as ribonucleases I, 11, and 111. The first peak is perhaps formed by an association of the already known ribonucleases with some acidic compound which becomes dissociated on Amberlite. Evidence is available in the case of crystalline beef (156) and mouse ribonucleases (154) that this compound may be a polynucleotide. All sheep ribonucleases I to IV have nearly the same specific activity and the same isoionic point which is very similar to the value found for beef ribonucleases A and B. The sequences in beef ribonuclease A and sheep ribonuclease I11 have been compared by the fingerprint technique (192). Only thrce specific differences are seen: substitution of a threonine by a serine a t position 3, substitution of a lysine by a glutamic acid a t position 37, and a still undetermined modification probably located between positions 99 and 104. Sulfuric acid, phosphate] and sucrose extracts of mouse and beef pancreas have also been compared chromatographically on Amberlite IRC-50 (154). For both species, the sulfuric acid extracts give two peaks which correspond to ribonucleases Aand B. When prepared from supernatant free of zymogen granules and microsomes, phosphate and sucrose preparations give a third peak, (10-15 % of the total activity in the case of mouse; 1% in the case of beef) which can be converted into the previous ones by acid treatment. This treatment induces a two- to three-fold increase in activity and the dissociation of the acidic compound already referred to above. The chemical structure of mouse ribonuclease(s) is still unknown.
PROTEINS OF EXOCRINE PANCREAS
19L
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83. Spackman, D. H., Stein, W. H., and Moore, S. (1958). Anal. Chem. SO, 1190. 84. Wilcox, P. E., Cohen, E., and Tan, W. (1957). J . Biol. Chem. 228,999. 85. Rovery, M., Charles, M., Guy, O., Guidoni, A., and Desnuelle, P. (1960). Bull. S O C . chim. biol. 42, 1235. 86. Rovery, M., Guy, O., Maroux, S., and Desnuelle, P. Unpublished experiments. 87. Kaasel, B., and Laskowski, M. (1961). J. Biol. Chem. 236,1996. 88. Smith, E. L., Brown, D. M., and Laskowski, M. (1951). J. Biol. Chem. 191, 639. 89. Oosterbaan, R. A., Kunst, P., Van Rotterdam, J., and Cohen, J. A. (1958). Biochim. et Biophys. Acta 27, 556. 90. Hartley, B. S. (1958). Biochem. J. 70, 4P. 91. Hartley, 13. S. (1958). Proc. Intern. Congr. Biochem., 4lh Congr., Vienna, Vol. 8, p. 87. V2. Hartley, B. S. (1959). J. Cellular Comp. Physiol. 64, 203. 93. Hartley, B. S. (1961). Proc. Intern. Congr. Biochem., bth Congr., Moscoiu (in press). 94. S h m , F., Keil, B., Holeyiiovskf, V., Meloun, B., Mikeb, O., and VanBbek, J. (1958). Collection Czechoslov. Chem. Commun. 23, 985. 95. Keil, B., and Sbrm, F. (1959). Collection Czechoslou. Chem. Commun. 24, 1558. 96. VanBEek, J., Keil, B., Meloun, B., and Sbrm, F. (1959). Collection Czechoslov. Chem. Commun. 24. 3148. 97. Meloun, B., Holeyiiovskf, V., VanGek, J., Keil, B., and S6rm, F. (1959). Colteclion Czechoslov. Cham.. Clom,mun 24.3002. 98. VanBbek, J., Meloun, B., Kostka, V., Keil, B., and Sbrm, F. (1960). Biochim. et Biophys. Acta 97, 169. 99. Tom&Bek, V., Holeybovskf, V., Mikeg, O., and Sbrm, F. (1960). Biochim. et Biophys. Acta 38. 570. 100. Meloun, B., VanBbek, J., Prusik, Z., Keil, B., and S6rm, F. (1960). Collection Czechoslov. Chem. Commun. 26, 571. 101. Keil, B., Sbrm, I?., HoleyRovskf, V., Kostka, V., Meloun, B., Mikeb, O . , Torn&Bek, V., and VanhEek, J. (1959). Collection Czechoslov. Chem. Commun. 24, 3491. 102. Kassel, B., and Laskowski, M. (1960). Federation Proc. 19,332. 103. Kassel, B., and Laskowski, M. (1957). Abstr. A m . Chem. SOC.1Slst Meeting, 6812. 104. Kassel, B. (1959). Federation Proc. 18, 257. 105. Rovery, M., Guy, O., and Desnuelle, P. (1960). Biochim. et Biophys. A d a 42,554. 106. Guy, 0. Unpublished experiments. 107. Gabeloteau, C., and Desnuelle, P. (1957). Arch. Biochem. Biophys. 69,475. 108. Gabeloteau, C., and Desnuelle, P. (1960). Biochim. et Biophys. A d a 42, 230. 109. Hofmann, T. (1960). Biochem. J. 74, 41P. 110. Hofmann, T. (1960). Biochem. J . 76, 26P. 111. Wootton, J. F., and Hess, G. P. (1958). Biochim. et Biophys. Acta 29, 435. 112. Viswanatha, T., Wong, R. C., and Liener, I. E. (1958). Biochim. et Biophys. A d a 29, 174. 113. Liener, I. E., and Viswanatha, T. (1959). Biochim. el Biophys. Acta 36. 250. 114. Tallan, H. H. (1958). Biochim. et Biophys. Acta 27, 407. 115. Liener, I. E. (1960). Arch. Biochem. Biophys. 88, 216. 116. Maroux, S., Rovery, M., and Desnuelle, P. Unpublished experiments. 117. Harris, J. I. (1959). Biochem. J. 71, 434. 118. Ravin, H. A., Bernstein, P., and Seligman, A. M. (1954). J. Biol. Chem. 208, 1. 119. Hofstee, B. H. J. (1957). Biochim. el Biophys. Acta 24, 211. 120. Martin, C., Golubow, J., and Axelrod, A. E. (1958). Biochim. et Biophys. Acta 27, 430.
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121. Martin, C., Golubow, J., and Axelrod, A. E. (1959). J. Biol. Chem. 234, 1718. 122. Inagami, T., and Sturtevant, J. M. (1960). J . B i d . Chem. 236, 1019. 123. Viswanatha, T., Lawson, W. B., and Witkop, B. (1960). Biochim. et Biophys. Acta 40,216. 124. Green, N. M., and Neurath, H. (1954). I n “The Proteins” (K. Bailey and H. Neurath, eds.), Vol. 11, Part B, p. 1057. Academic Press, New York. 125. Neurath, H., and Dreyer, W. J. (1955). Discussions Faraday SOC.No. 20, 32. 126. Charles, M., Rovery, M., Maroux, S., and Desnuelle, P. Unpublished experiments. 127. Anson, M. L. (1935). Science 81,467. 128. Neurath, H. (1960). I n “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), 2nd ed., Vol. IV, p. 11. Academic Press, New York. 129. Neurath, H., Brown, J. R., Greenshields, R. N., Keller, P. J., and Yamasaki, M. (1961). Federation Proc. 20, 220. 130. Thompson, E. 0. P. (1953). Biochim. et Biophys. Acta 10. 633. 131. Ando, T., Fujioka, H., and Kawanishi, Y. (1959). Biochim. et Biophys. Acta 34, 296. 132. Vallee, B. L., Rupley, J. A., Coombs, T. L., and Neurath, H. (1958). J. A m . Chem. Soc. 80,4750. 133. Vallee, B. L., Rupley, J. A., Coombs, T. L., and Neurath, H. (1960). J . B i d . Chem. 236, 64. 134. Coleman, J. E., and Vallee, B. L. (1960). J. Biol. Chem. 236, 390. 135. Vallee, B. L., Coombs, T. L., and Hoch, F. L. (1960). J. B i d . Chem. 236, PC45. 136. Coleman, J. E., and Vallee, B. L. (1961): Federation Proc. 20, 220. 137. Folk, J. E., and Gladner, J. A. (1960). J. Biol. Chem. 236, 60. 138. Waldschmidt-Leitz, E., Ziegler, F., Schaffner, A., and Weil, L. (1931). 2.physiol. Chem. Hoppe Seyler’s 197, 219. 139. Weil, L., Seibles, T. S., and Telka, M. (1959). Arch. Biochem. Biophys. 79, 44. 140. Folk, J. E., and Gladner, J. A. (1961). Biochim. et Biophys. Acta 48, 139. 141. Smith, E. L., and Stockell, A. (1954). J. B i d . Chem. 207, 501. 142. Desnuelle, P. Advances i n Enzymol. 23,129. 143. Sarda, L., Marchis-Mouren, G., Constantin, M. J., and Desnuelle, P. (1957). Biochim. el Biophys. Acta 23, 264. 144. Marchis-Mouren, G., Constantin, M. J., and Desnuelle, P. (1958). Bull. SOC. chim. biol. 40, 2019. 145. Sarda, L., Marchis-Mouren, G., and Desnuelle, P. (1957). Biochim. el Biophys. Acta 24, 425. 146. Sarda, L., and Desnuelle, P. (1958). Biochim. et Biophys. Acta 30, 513. 147. Sarda, L., PasBro, L., and Desnuelle, P. Unpublished experiments. 148. Sarda, L., Benzonana, G., and Desnuelle, P. Unpublished experiments. 149. Desnuelle, P., Sarda, L., and Ailhaud, G. (1960). Biochim. et Biophys. Acta 37, 570. 150. Laskowski, M. (1951). In “The Enzymes” (J. B. Sumner and K. Myrback, eds.), Vol. I, Part 2, p. 956. Academic Press, New York. 151. Anfinsen, C. B., and White, F. H., Jr. (1961). In “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. V, p. 95. Academic Press, New York. 152. Martin, A. J. P., and Porter, R. R. (1951). Biochem. J . 49, 215. 153. Hirs, C. H. W., Moore, S., and Stein, W. H. (1953). J. Biol. Chem. 200. 493. 154. Dickman, S. R., Morrill, G. A., and Trupin, K. M. (1960). J. B i d . Chem. 236,169. 155. Iqvist, S. E. G., and Anfinsen, C. B. (1959). J. Biol. Chem. 234, 1112. 156. Taborsky, G. (1959). J. B i d . Chem. 234, 2652.
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ENZYME FRACTIONATION BY SALTING-OUT: A THEORETICAL NOTE By MALCOLM DIXON and EDWlN C. WEBB Department of Biochemistry, University o f Cambridge, England
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I. Introduction..
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197 198 198 2Q7 211 211 213
B. Mixtures of Proteins.. ................................................ 111. Application of Theory to Protein Fractionation.. ......................... A. Effect of Protein Concentration on Fractionation.. .................... B. Optimum Fraction Limits. ............................................ C. Successive Fractionations. . . . . . . . . . . . . . . . . . . . . . . . . . . 214 D. Choice of Salt.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 IV. Conclusions Relating to Enzyme Fractionation. .......................... 217 218 References.. .............................................................
I. INTRODUCTION For many years salting-out by high concentrations of ammonium sulfate has been one of the classical methods of protein separation. There is very little literature on the theoretical basis of the method, particularly as applied to the isolation of enzymes, where it has mainly been used quite empirically. The underlying assumption in most cases seems to have been that the different proteins are precipitated at different fixed ammonium sulfate concentrations, provided the pH and temperature are fixed. For example one may commonly read in instructions for the purification of an enzyme that “the enzyme is precipitated at 65% saturation with ammonium sulfate” or that “the fraction precipitating between 0.62 and 0.68 saturation should be taken.” It is, however, a fairly common experience that when one repeats a published method the enzyme fails to precipitate within the limits given. Furthermore, where the purification of a protein involves more than one salt-fractionation stage, the limits are usually found to be different for the different stages. We shall show that the idea that a given protein will always precipitate at a fixed concentration of electrolyte is quite erroneous. We hope that the following theoretical treatment will facilitate the more precise use of the method. 197
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11. THEORY OF SALTING-OUT A . One Protein The effect of various factors on the solubility of a protein in salt solutions has been discussed by a number of authors, e.g., Cohn and Edsall (1943), Green and Hughes (1955), Czok and Bucher (1961). If one progressively adds electrolyte to a protein solution, two effects are observed as the ionic strength increases. There is first an increase in solubility, due to a decrease in the activity coefficient of the protein (Scatchard and Kirkwood, 1932). In higher electrolyte concentrations the second effect, causing a diminished solubility, becomes dominant, so that the solubility passes through a maximum. Figure 1 shows these two effects, known as salting-in and salting-out, respectively. In the salting-in process it is usually the cations which are important, except at very low ionic strength, and salts with multivalent cations which are most effective. A theoretical treatment of the effect of electrolyte on activity coefficients of proteins is difficult, but an expression has been developed by Kirkwood (1934) on the basis of certain simplifying assumptions. We shall not concern ourselves with this region, since the theory of salting-in, and the experimental findings, have been extensively reviewed elsewhere (Cohn and Edsall, 1943; Edsall and Wyman, 1958). In the salting-out region it is the anions which are important, although the nature of the cation may have a secondary influence. Salts with multivalent anions, such as sulfates, phosphates, and citrates, are especially effective; salts such as sodium and potassium chlorides are relatively ineffective. It will be seen from Fig. 1 that in the salting-out region the graph of the logarithm of the solubility of a protein against the ionic strength approximates to a straight line. Expressed in mathematical terms (Cohn, 1925) this becomes
where S is the solubility of the protein in grams per kilogram of water, 1/2 is the ionic strength in moles per kilogram of water, and @ and Ksare constants. It is more convenient to express the concentration terms per liter of solution, and the equation then becomes log s
=
6'
- K sr r
with different constants @' and KL . Here s is the solubility of the protein in grams per liter of solution and r/2 is the ionic strength in moles per liter of solution. Our discussions will be in terms of Eq. (1).
ENZYME FRACTIONATION BY SALTING-OUT
199
The constant Kfi represents the slope of the graph, and the constant 0' is given by the point of intersection of the straight line with the vertical axis and represents the logarithm of a hypothetical solubility a t zero electrolyte concentration.
-1.510
1 I
I
1
1
I
2
3
4
5
r/2
1 6
FIG.1. Relation between log s and ionic strength for carboxyhemoglobin and ammonium sulfate. Plotted from data of Green (1931,1932). The pH is 6.6; temperature, 25°C; s is given in grams of protein per liter; and the ionic strength of ammonium sulfate in moles per liter. SO= 17 =/liter.
Salting-out equations of this form are not peculiar to proteins; similar equations are obeyed by most organic substances and even by dissolved gases, which may be salted-out by electrolytes. The cause is not wholly understood; one suggestion is that the salt ions, in becoming hydrated, remove part of the water, which is therefore unavailable as a solvent.
200
DIXON AND WEBB
Figure 2 shows solubility curves for a number of proteins in ammonium sulfate solutions. The number of proteins that have been studied in this way is small, and in even fewer cases has a study of tjhe effect, of variables such as pH and temperature been carried out,. From these studies, however, certain generalizations can be deduced.
r
FIQ.2. Relation between log s and ionic strength of ammonium sulfate for several proteins. From Cohn and Edsall (1943). F = fibrinogen; COHb = carboxyhemoglobin; PG = pseudoglobulin; SA = serum albumin; COMb = carboxymyoglobin. TABLE I 8' and KL f o r Carboxyhemoglobin for Various Salts Constant
Potassium phosphate
Sodium sulfate
Ammonium sulfate
Sodium citrate
Magnesium sulfate
B' K',
3.01
2.53 0.76
3.09 0.71
2.60 0.69
3.23 0.33
1 .oo
KL is independent of pH and temperature, but depends on the nature of the protein and of the salt. Even so, these variations are not enormous; with ammonium sulfate the variation of K i with different proteins does not appear to be more than twofold. With the same protein and different salts, KL decreases in the following order : potassium phosphate, sodium sulfate, ammonium sulfate, sodium citrate, magnesium sulfate (Table I ) . For chlorides the value is very much lower.
ENZYME FRACTIONATION BY SALTINQ-OUT
20 1
P' on the other hand varies markedly with the nature of the protein, but is more or less independent of t,he nature of the salt, as would be expected since it is obtained from an extrapolation to zero ionic strength. It is the value of 0' which chiefly determines the solubility of a protein in strong salt solutions. P' is dependent on both p H and temperature. Figure 3 shows the variation of P' with p H for ovalbumin and carboxyhemoglobin. Changes of well over 1 unit per unit of pH are frequently
u OA
COHb
4
O3
4
5
6
7
8
PH
FIQ.3. Dependence of 8' on pH. Plotted from data of Green (1931) for saltingout with phosphate (COHb) and ammonium sulfate (OA). OA = ovalbumin; COHb = carboxyhemoglobin.
encountered, and bearing in mind that P' represents the logarithm of a solubility, we see that this corresponds to a change of over tenfold in the solubility per pH unit. P' usually passes through a minimum in the neighborhood of the isoelectric point, and in this region depends little on pH. More than one minimum may exist: for example, horse carboxyhemoglobin shows two minima (Sgrensen and Sgrensen, 193:3), one a t pH 6.6, corresponding to the isoelectric point of the protein, and one at p H 5.4, which is said to be due to the formation of a less soluble carboxyhemoglobin sulfate. The relative solubilities of two proteins may vary very rapidly with pH; for example, it will be seen from Fig. 3 that a change of pH from 5 to 6 would
202
DIXON AND WEBB
change the ratio of the solubilities of the two proteins at a given salt concentration by several thousand times. A rise in temperature commonly causes a decrease in pi, although the opposite effect is also found. I n the case of ovalbumin the solubility has a minimum value at 25°C (Sgrensen and Hgyrup, 1915-1917). An ammonium sulfate solution saturated with a protein at 0°C may throw out 90% of this protein if the solution is allowed to warm up to room temperature. For example, carboxyhemoglobin is ten times as soluble in ammonium
2
25.C
pH 6.6
“ I
-
0 0
PH 7.43
0
7.17 6.05 I
3
r/2
I 4
6.80 ‘6.60
3
1-12
. 4
FIQ.4. Effect of temperature and pH on salting-out curves of carboxyhemoglobin by phosphate. Replott,ed from Green (1931).
sulfate at 0°C as it is at 25”C, and myosin is even more sensitive to temperature. One of the best methods of crystallizing many proteins from ammonium sulfate is to allow the temperature to rise slowly. Since the effects of a change of temperature are different with different proteins, the order of precipitation of a given series of proteins as the salt concentration is increased may be quite different at different temperatures. Figure 4 shows the effect of change of pH and temperature on the log 8 curve for carboxyhemoglobin (in this case for phosphate) (replotted from Green, 1931). It will be Seen that the curves are parallel, showing the constancy of KL . The position of the line in relation to the vertical scale varies because of the variation of p i ; since it is straight, the same effect could be produced by a displacement along the horizontal axis. In other words a
ENZYME FRACTIONATION BY SALTING-OUT
203
certain increase in 0’ means that the ionic strength must be increased by a certain amount to give the same solubility of the protein. The proportionality constant will be determined by Ks, and with an average value of the latter it can be calculated that an increase of 1 unit in @’ corresponds to an increase of very roughly 7 in the percentage saturation with ammonium sulfate. I n other words if @’ is increased by 1, one would need about 67% saturation to produce the same effect as was previously produced by 60 % saturation. A change of this order may sometimes be produced by a change of pH of as little as 1 unit or of temperature by 25°C. Clearly pH and temperature are two variables that should be carefully controlled while carrying out salt fractionations. Insufficient attention has been given to this in the past; in published methods of enzyme isolation, while the pH is often stated, the temperature is more often omitted.’ Now let us assume that we have a solution of a single protein of initial concentration Co grams per liter, and that we gradually increase the percentage saturation with ammonium sulfate, say by steps of 1, removing the protein that is precipitated each time, We thus get a series of fractions corresponding to the ranges . . . 60-61, 61-62, 62-63 . . . % saturation with salt. How will the protein be distributed between the fractions? For calculating the distribution, the usual log s curves (Fig. 2) are not very convenient, and curves of actual solubility are needed. Figure 5 shows such curves, plotted from Fig. 2, giving solubility against percentage saturation with ammonium sulfate ( P ) . In the region of protein precipitation, the slope of these curves determines the amount of protein found in each fraction. It is therefore convenient to plot the slopes of these curves (i.e., -ds/dP) against P, as shown in Fig. 6. It will be seen that there is a close similarity between the curves of Fig. 5 and the corresponding curves of Fig. 6. In fact for any given protein the curves of Fig. 5 and 6 are identical, apart from a difference of scale in the vertical direction. This can be shown mathematically as follows. Differentiating Eq. (1) we obtain
Since dln 8 = ds/s, ds = 2.303 X s X d ( r / 2 )
-&
1 After the manuscript of this article had been submitted for publication, the Editors informed us of the existence of the review of Czok and Bucher (1961),which was then in proof, and drew our attention to the fact that this included some of the material given in the preceding parts of this section. We have, however, left them unchanged, as they form an essential part of the present discussion.
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DIXON AND WEBB
so that
ds/d( r/2) = -2.303 KL.s
(2) Ignoring any contribution of the protein itself to the ionic strength, as is customary, r/2 is proportional to the percentage saturation P a t a fixed temperature. Therefore we may write Eq. (2) as
ds/dP = K . s where K is approximately equal to -2.303
(3)
x
KL
x
8.0, the factor 8.0
FIG.5. Some solubility curves obtained from Fig. 2
being the increase of P corresponding to an increase in I’/2 of 1.0 a t room temperature. I n other words, a t any salt concentration the height of the differential curve for any protein is proportional to the height of the solubility curve. The slight differences between Figs. 5 and 6 are due to the fact that K; has slightly different values for the different proteins, giving different, scale ratios. Returning to the postulated solution containing a single protein, it is obvious that as the salt concentration is increased no precipitation of protein will oceur until s falls to Co . At that point precipitation will suddenly start; from the form of the curves in Fig. 6 it will be seen that precipitation
ENZYME FRACTIONATION BY SALTING-OUT
205
will start at a high rate and fall off in successive fractions; once precipitation has begun, one is effectively running down the differential curve. The distribution curve will therefore be a peak with a sharp edge on the side of lower salt concentration, with an asymptotic tailing off on the other side (see, for example, Fig. 7). The width of the peak is determined only by K i and is independent of j3’, so that it does not vary very greatly from one protein to another. The actual position of the peak in a horizontal direction depends also on /3’ and on the concentration of the protein.
FIG.6. Rate of change of solubility with ammonium sulfate concentration, obtained by differentiating curves of Fig. 5.
For a given protein, the distribution curves for different values of COare all determined by the same differential curve. Co merely determines the point of entry to the curve. Taking the case of two solutions of carboxymyoglobin, one containing 30 gm per liter and the other being a tenfold dilution of this, we read off from Fig. 5 the respective salt concentrations for first precipitation (points A and B) and transfer them to Fig. G. The two distribution curves of the precipitation will now be portions A’ to C and B’ to C of the differential curve in Fig. G for the stronger and weaker solutions respectively. The two peaks represented by the areas AA’C and BB’C are of course of different sizes because of the different amounts of protein taken, but if they are normalized by expressing the amounts as
206
DIXON AND WEBB
3gm/liter
30 gm/litrr
8-
r6-
5-
4-
3-
2-
i-
"
40
50 60 %Saturation with ammonium sulfate /PI
Fro. 7. Distribution curves for salting-out of carboxymyoglobin a t two concentrations, obtained from curves of Fig. 6.
percentages of the original amount of protein taken, one obtains the curves shown in Fig. 7. The two normalized curves are identical in form, but occur at different salt concentrations.' 2
This may be shown as follows. From Eq. ( 2 ) , writing D for - d s / d ( r / 2 ) , we have
D
2.303 Kk.8
From Eq. (l), log s = 8' whence log D Multiplication of
-
- K;(r/2)
log 2.303 KA
D by a factor
a gives log aD = log 2.303 KJ
+ 8' - K ; ( r / 2 )
+ 8' - K i
(a x) - log a
(4)
Thus by multiplying all the ordinates by a factor, as has been done for the right hand
ENZYME FRACTIONATION BY SALTINQ-OUT
207
This has a very important practical consequence. From a carboxymyoglobin solution containing 30 gm per liter the greater part of the protein will be precipitated between 58 and 65% saturation with ammonium sulfate. From the solution which has been diluted ten times, no protein whatever will be precipitated within this range; precipitation will begin only at 66%, and the corresponding precipitation limits will be 66 to 73% saturation. In other words, contrary to widespread and long-standing belief, proteins do not appear in fractions between fixed characteristic limits of ammonium sulfate concentration, but on the contrary the fractionation limits vary with the concentration of the particular protein.
B . Mixtures of Proteins In the absence of interaction between proteins, it would be expected that the different proteins of a mixture would precipitate independently in accordance with the theory given in the previous section. It is often assumed that a precipitating protein will tend to bring down a certain amount of the other proteins with it, so that the range of precipitation of any one protein will be dependent on the other proteins present and a sharp separation is prevented. It is well known that under certain conditions interaction between different proteins can be observed. However, we believe that this has never been shown in presence of a high concentration of electrolytes. The surface potential is no doubt the main factor in determining such interaction, and it is well known that this is reduced to very low values at high salt concentrations, such as are present in the salting-out region. This is easily seen with adsorbents: ammonium sulfate solutions are very effective in eluting proteins from even powerful adsorbents. Positive evidence that the proteins of a mixture precipitate independently during fractional salting-out is provided by the curves given later. Let us suppose that we have a solution containing two proteins which do not interact in the presence of salt, and that we precipitate with gradually increasing concentrations of ammonium sulfate. To take an actual case, let us assume that we have equal concentrations of serum albumin and carboxymyoglobin (Fig. 8a and b ) . As the solubility curves for these proteins are well separated, the precipitation will occur in two distinct steps (Fig. ~~
~~
curve of Fig. 7, and decreasing r/2 by an amount (log a ) / K i , the original curve is obtained. This means that the effect of diluting the protein solution ten times is to increase the ionic strength required for precipitation by an amount corresponding to (log a ) / K i . Taking the value of K i for carboxymyoglobin as 1.1, this increase is approximately 0.9, which is equivalent to an increase of about 7 in the value of P,as shown in Fig. 7. The width of any peak a t half the maximum height is (log 2 ) / K i , and is therefore inversely proportional to K i . If K i = 1,the width is 0.3ionic strength units, which corresponds t o a range of 2.4 in per cent saturation with ammonium sulfate.
1
lo)
ICl
80
c
.-
e
0
z20c
z
I
30
40
50
I
5-
4-
-llL 0 2
30
40
50
--dS dP 3-
2-
60
70 30 40 %Saturation with ammonium sulfate (PJ
I-
IN I
5
c
w
I i
50
FIG.8. Solubility and distribution curves for pairs of proteins. (a), (b) Serum albumin and carboxymyoglobin, each at 30 gm/liter. (c), ( d ) Pseudoglobulin and serum albumin, each at 30 -/liter. ( e ) , 0, (9) Pseudoglobulin and serum albumin, each at 10 gm/liter. ( a ) , (c), ( e ) show total protein concentration in solution. ( b ) , ( d ) , cf) show distribution curves of the individual proteins in the piecipitate. (gj shom a distribution curve of total protein precipitated
ENZYME FRACTIONATION BY SALTING-OUT
209
8b) ; the precipitation of the serum albumin is virtually complete before that of the carboxymyoglobin begins. Each protein gives a distribution curve with a sharp peak of the form discussed in the last section. If one plots the concentration of total protein remaining in solution against salt concentration, one obtains the curve in Fig. 8a, in which the position of each peak in Fig. 8b is shown by a sharp drop. It will be noticed that the shapes of the curves for the two proteins are not quite identical because these two proteins have different values of KL . Such a clear separation of two proteins will not always be obtained. To go t o the other extreme, Fig. 8c and d shows the corresponding curves for a solution containing serum albumin and pseudoglobulin, each a t 30 gm per liter. The precipitation peaks occur a t the same salt concentration, and therefore the residual protein curve shows a single smooth step. If the same mixture is diluted three times, the curves shown in Fig. 8e and f are obtained. Both peaks have been moved to the right by the dilution, but by different amounts, due to the different values of KL for the two proteins. The residual protein curve now shows two discontinuities, although the precipitation of the pseudoglobulin is by no means complctr a t the point where the precipitation of serum albumin begins. Figurc 88 shows the actual amount of protein precipitated, i.e., the sum of the separate curves of Fig. 8f, or in other words the differential curve corresponding to Fig. 8e. Although it was not possible to separate the two proteins at the higher concentration, after dilution more than half of the pseudoglobulin can be obtained in the pure state between the limits 33 and 36 % saturation with ammonium sulfate. The curves of Fig. 8 are theoretical, calculated from the known solubility constants of the proteins. Roche and Derrien (1946) have published actual curves of solubility in ammonium sulfate of an artificial mixture of pure crystalline serum albumin (horse) and hemoglobin (dog), and have compared them with the corresponding curves for the separate proteins (Fig. 9). It will be seen that the correspondence is very close. With a mixture of several proteins, in the absence of interaction, each protein should be represented by a definite discontinuity in the residual protein curve, provided that the points of first precipitation of no two proteins coincide. This is what is found in practice; examples are shown in Figs. 10 and 11. Figure 10, from Butler et al. (1935), shows curves for the precipitat,ion of horse plasma proteins by phosphate a t three different pH values. It will be seen that a number of proteins are precipitating indcpendently, the points of first precipitation being markedly pH-dependent . Figure 11, from Falconer et al. (1953), shows some more recent curves for precipitation of rat liver proteins by salting-out with ammonium sulfate. Parallel estimations of the light absorption at 405 mp by the remaining
210
DIXON AND WEBB
solution are also shown in the figure, and give an index of the amount of hemoproteins in solution. It will be seen that not all the proteins are hemoproteins; although the second and the last proteins to precipitate are clearly in this category. The fact that their precipitation begins suddenly is further evidence of the absence of interaction of the type mentioned at the beginning of this section. 0.8I-
FIQ.9. Solubility curvea for pure crystalline serum albumin, for hemoglobin, and for a mixture of the two, shown as a function of ammonium sulfate concentration. From Roche and Derrien (1946). 0 = hemoglobin (dog); 0 = serum albumin (horse); X = mixture of both. The curve for hemoglobin has been displaced upwards (scale on right) to show the close correspondence with the curve for the mixture.
Figure 12 shows the distribution curve obtained by differentiation of curve A of Fig. 11. Each protein is represented by a separate peak. Because the curve for each protein falls asymptotically to zero, each protein will contaminate all the other fractions which come after, but the amount of contamination will fall off rapidly with increasing separation. On the other hand, no one protein will contaminate fractions on the left of its peak (except of course in so far as some supernatant may be mechanically trapped in the precipitate). Only the first protein to be precipitated will be obtained 100% pure, although later proteins may be obtained in a high degree of purity if the peaks are well separated.
ENZYME FRACTIONATION BY SALTINQ-OUT
21 1
111. APPLICATION OF THEORY TO PROTEIN FRACTIONATION
A . Efect of Protein Concentration on Fractionation It has been said that to obtain good separation of proteins by salt fractionation it is advantageous to use a high concentration of protein. The efficiency of separation is determined by the width of the peaks in the distribution curve and their separation. The theory indicates that the width of a given peak is independent of the protein concentration (see footnote 2).
FIG.10. Curves showing residual dissolved protein for salting-out of horse plasma proteins with phosphate at three pH values. From Butler et al. (1935).
The effect of the concentration of any protein on the position of its peak is determined by the value of KL for that protein. Therefore in a mixture of proteins with different K[,values the relative positions of the various peaks will vary with the concentration of the mixture, as pointed out by Falconer et al. (1953). The effect of this on the efficiency of the fractionation is unpredictable unless the values of KL are known. The effect of diluting a mixture may be to separate two peaks that were previously overlapping, as in the case considered in Fig. 8c to g; on the other hand dilution may bring together peaks that were previously well separated. Protein concentration is clearly a variable that should be taken into account in finding the best conditions for the isolation of any particular protein, and it may be worth
212
DIXON AND WEBB
while to carry out successive fractionations at different protein concentrations. Excessive dilution, however, should be avoided for three reasons. Since dilution shifts all the peaks to higher concentrations, an increasing propor-
%Saturation wilh ammonium sulfate fP)
FIG.11. Precipitation of soluble rat liver proteins with ammonium sulfate. Redrawn from Falconer et al. (1953). Curve A (scale on left): protein remaining in solution. Curve B (scale on right) : absorbance of supernatant solution at 405 mp.
tion of the proteins will be left in solution when the saturation limit of the salt is reached, and everitiially whole peaks may be moved beyond this limit. Furthermore protcin precipitatrs produccd at high ammonium sulfate concentrations are difficult to pack proptdy hy centrifugation arid filtration must hc resorted to. The third point, which may be of importanre in large scale work, is one of expense; dilute solutions require considerably more ammonium sulfate to achieve the same result.
ENZYME FRACTIONATION BY SALTING-OUT
213
B. Optimum Fraction Limits The theory can be used to throw light on the size of step which should be chosen in isolating a protein from a mixture. It is not infrequent to read of the use of very wide fraction limits in enzyme preparations, e.g., from
0.20 O ’ t
--d f dS
0.15
0.10
0.05
I
I
I
25 50 %Saturation with ammonium sulfate
(PI
75
FIG.12. Distribution curve of total protein precipitated, plotted from curve A of Fig. 11 by graphical differentiation (approximate only). Ordinates expressed in terms of grams of protein per liter.
45 t o 65% saturation. Figure 7 shows, however, that about 90% of the
precipitate occurs within a range of 7 4 % of saturation with ammonium sulfate, and although this varies somewhat for proteins with different K; values, it does not vary very greatly and indicates the order of fraction limits needed to precipitate the bulk of any particular protein. If the protein is contaminated by overlap of the “tail” of a preceding peak, one might suppose at first sight that the purest fractions would occur
2 14
DIXON AND WEBB
at the highest part of the peak, with the purity falling off as the salt concentration is increased. However, if the two proteins have the same value of KL this is not the case. It follows from the fact that both curves are logarithmic, with the same proportionality constant, that the ratio of the ordinates of the two curves is independent of the abscissa; in other words, once precipitation of both proteins is proceeding, the ratio of the proteins in the precipitate is independent of the salt concentration. This is only true if the two Kh values are the same; Fig. 8d shows a case where they are different. According to the relative values of K i for the two proteins, the purity may vary in either direction. However, normally any change in purity will not be very great. To get the maximum yield with a given degree of purification, the fraction should extend from one peak almost to the next. Normally, of course, the positions of the peaks are not known, except that in purifying an enzyme the point of first precipitation can be determined by activity tests. Then, in the absence of any knowledge about the precipitation of the other proteins, it is probably best to take a range which precipitates about 75% of the enzyme concerned. As we have shown, this will normally correspond to an increase of 6 % or thereabouts in saturation.
C.Successive Fractionations The question now arises whether a repetition of the fractionation under the same conditions is likely to effect a further purification of a given protein, e.g. an enzyme. In considering the other proteins which are contaminating the enzyme, three cases must be distinguished, namely those whose peaks lie on the right-hand side of the fraction considered, those whose peaks lie on the left of the fraction, and those whose peaks lie within the enzyme fraction (Fig. 13a). Suppose for simplicity we take the fraction precipitated within the range shown, and dissolve it in the same volume as before, and reprecipitate taking the same fraction limits (Fig. 13b). The proteins (3 and 4) precipitating on the right of the fraction in Fig. 13a will not appear in that fraction. As far as these proteins are concerned, nothing is to be gained by the repetition. The proteins on the left (e.g. 1 ) will mainly have been removed and rejected before obtaining the enzyme fraction; however, a part of these proteins will be present in the second fractionation, and the fact that much of these proteins has been discarded will not affect their concentration at the initial salt concentration of the enzyme fraction. The enzyme fraction therefore starts the second time with the same concentration of these contaminating proteins as it did the first time. So far as these proteins also are concerned, therefore, there will be no improvement on refractionation. The situation is different with regard to those impurities whose peaks lie within the chosen fraction limits, particularly those with peaks near
ENZYME FRACTIONATION BY SALTING-OUT
215
the right-hand limit. Only part of such a protein (e.g. 2) will have been precipitated with the fraction on the first occasion, so that refractionation will start with a lower concentration of this protein. Thus its peak will be
4
First fractionation
U Fraction taken
P
E Second fractionotion
--
ds dP j
.U Fraction taken
P
Fra. 13. Imaginary curves to illustrate the effect of refractionation in purification of an enzyme (E). See text for description.
shifted to the right and may even be shifted outside the fraction altogether. In any case the proportion of thie protein precipitating with the enzyme will be reduced by the repetition. Thus repetition is only beneficial with respect to the thiid group of proteins. The situation is more complicated if, as is usually the case, the refractionation is carried out from a smaller volume than the first fractionation;
216
DIXON AND WEBB
if for example the total protein concentration is held more or less constant by taking up the precipitated fraction in a correspondingly smaller volume. The concentration of the enzyme and proteins precipitating close to it will now be higher, while that of other contaminating proteins may be considerably lower. The main effect of this is that the fraction limits must be moved towards lower salt concentrations to obtain the same recovery of enzyme. The peaks of the main contaminants will move in the same direction, but as explained in Section 111, A they may not all move to the same extent. The separation of the peaks, and therefore the purification, may be either incfeased or decreased compared with the case of Fig. 13b according to the KSvalues concerned. The general conclusion is that a repetition under the same conditions is of very limited value, and that one can do much better by repeating the fractionation under different conditions. In particular, in Section 11, A attention was drawn to the great value of change of pH and temperature in altering the fractionation pattern. The potentialities of alterations of temperature have scarcely been realized at all. Even in the case of pH, the possible value of changes of pH between successive fractionations has not been fully appreciated.
D . Choice of Salt A salt that is to be suitable for protein fractionations must be highly soluble, reasonably cheap, and without any direct effect on proteins. Ammonium sulfate has been most commonly used in enzyme fractionations, as it fulfills these requirements. In addition, as shown in Table I, it gives reasonably high KL values for proteins. Disadvantages are that pure ammonium sulfate is slightly acid and many commercial samples highly so, that it is not a buffer and pH control is difficult owing to loss of ammonia, and that nitrogen estimations cannot be done without careful removal of the salt. It is not easy to determine the pH of strong ammonium sulfate solutions because of salt errors and junction potentials; the salt should be diluted to less than 0.3 M before determination is attempted. In using high concentrations of salts, allowance must be made for the fact that there are considerable volume changes when amounts of salt of the required order are dissolved in water, or when saturated solutions are diluted with water. Knowledge of these changes is necessary for the calculation of the concentration. In the case of ammonium sulfate, a nomograph is available (Dixon, 1953) which avoids much troublesome calculation. Considering other salts that can be used, we note that chlorides are extremely inefficient. Sodium sulfate is rather more effective than ammonium sulfate, but much less soluble; it can therefore only be used for certain proteins. Magnesium sulfate is less effective, giving low values of Kh (Table I) ;
ENZYME FRACTIONATION BY SALTING-OUT
217
therefore the peaks are less sharp. Phosphates, however, work very well; they give high KL values, and therefore sharp peaks and very good separations. IJurthermore they are buffers in the most useful range of pH, so that the pH during the fractionation call be acciirately controlled without difficulty. They do not interfere with nitrogen estimations and are quite soluble, provided that the right cations are chosen. In neutral and alkaline solutions, potassium phosphate buffers of high concentration can be made; in acid solution sodium phosphate buffers are more soluble. Even with phosphates, care must be taken with the control of pH; the pH depends appreciably on the ionic st>rength,and at high ionic strength sodium phosphate buffers are much more acid than potassium phosphate buffers of the same molecular composition.
IV. CONCLUSIONS RELATING TO ENZYME FRACTIONATION Nothing that has been said in the previous sections should be taken as disparaging salt fractionation as a method of enzyme purification. It is in fact a method with a great many advantages. It is easy to carry out without expensive materials or equipment. Unlike fractionation with organic solvents, it does not tend to inactivate enzymes by denaturation; on the contrary, ammonium sulfate frequently has a protective action on enzymes, and indeed enzymes are frequently stored in the form of suspensions of precipitates in concentrated ammonium sulfate. This not only stabilizes the enzyme but prevents the growth of bacteria. Unlike chromatographic and adsorption methods, it affords a procedure for concentrating as well as purifying the enzyme. Perhaps the most important consequence of the preceding discussion is that enzymes do not come down within fixed limits of salt concentration characteristic of each enzyme, but that the limits vary with the concentration of the enzyme. Fixed characteristic limits can be obtained, however, if the initial solution is always diluted to the same enzyme activity. Unless this is done, the precipitation limits of the enzyme will vary, not only with the stage of purification, but also with the activity of the starting material. Thus in following a published method for purification of an enzyme from yeast, say, one may find that the limits must be modified because the sample of yeast available may have a different content of the enzyme. We have stressed the importance of pH and temperature as variables that have a great effect on the positions and separation of the precipitation peaks. They must therefore be carefully controlled. Furthermore, by varying them in accordance with the principles stated above, it should be possible to increase greatly the versatility and effectiveness of the fractionation. Temperature is not always easy to control. It is fairly common practice
218
DIXON AND WEBB
to store a large volume of extract in the cold and then transfer it to the laboratory for fractionation. The large volume will only warm up comparatively slowly, and the different fractions will then be obtained at different temperatures, which are rarely recorded. Variation of temperature introduces a difficulty in the use of percentage saturation as a measure of salt concentration, since the solubility of the salt will vary with temperature. Some data for ammonium sulfate are given by Taylor (1953). It would be preferable always to state the actual concentration of salt, for example as molarity. A survey of the literature of enzyme purification suggests that there has been uncertainty as to the most satisfactory salt concentration increment to use at each step. The theory that we have discussed provides some guidance on this point. It would appear that a span of from 5 to 10 in the percentage saturation with ammonium sulfate, if properly chosen, should provide the best compromise between good purification and good yield. It also appears that repetition of salt fractionation under the same conditions is unlikely to be very useful, but that repetition under differen$ conditions may contribute a considerable further purification. Throughout the above discussion we have assumed that at each stage the solution is in equilibrium with a single form of each protein precipitated. I n salting-out procedures it may take an appreciable time to attain this condition, and if the precipitates are removed too soon the results may diverge from those predicted by the foregoing theory. On the other hand, if the mixture is left for a much longer time (e.g. several days) crystallization may occur, introducing a new complication. A single protein may crystallize in several different forms with different solubilities, and the solubility, and hence the composition of the precipitate, may change in a complicated manner with time. This point has been stressed by Czok and Bucher (1961).
REFERENCES Butler, A. M., Blatt, H., and Southgate, H. (1936). J . Bio2. Chern. 109, 756. Cohn, E. J. (1926). Physiol. Revs. 6, 349. Cohn, E. J., and Edsdl, J. T. (1943). “Proteins, Amino Acids and Peptides.” Reinhold, New York. CBok, R., and Biicher, T. (1961). Advances in Protein Chem. 16, 316. Dixon, M. (1963). Biochem. J . 64, 468. Edsall, J. T., and Wyman, J. (1968). “Biophysical Chemistry,” Vol. I, Chapter 5. Academic Press, New York. Falconer, J. S.,Jenden, D. J., and Taylor, D. B. (1963). Discussions Faraday Soc., No. 18, 40. Green, A. A. (1931). J . Biol. Chem. B8, 496, 617. Green, A. A. (1932). J . Biol. Chem. 96, 47. Green, A. A., and Hughes, W. L. (1966). I n “Methods in Enzymology,” (S. P. Colowick and N. Kaplan, eds.), Vol. I, p. 87. Academic Press, New York.
ENZYME FRACTIONATION BY SALTING-OUT
219
Kirkwood, J. G . (1934). J . Chem. Phys. 2, 351. Roche, J., and Derrien, Y. (1946). Bull. S O C . chim. biol. 28, 838. Scatchard, G . , and Kirkwood, J. G. (1932). Physik. 2. 33, 297. Sorensen, S . P. L., and Hgiyrup, M. (1915-1917). Compt. rend. trav. lab. Carlsberg, SBr. chim. 12, 213. Sfiremen, S. P. L., and SZrensen, M. (1933). Biochem. Z . 268, 16. Taylor, J. F. (1953). In “The Proteins,” (H. Neurath and K. Bailey, eds.), Vol. I, Part A, p. 2. Academic Press, New York.
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NONENZYMATIC METHODS FOR THE PREFERENTIAL AND SELECTIVE CLEAVAGE AND MODIFICATION OF PROTEINS By 8. Witkop laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland
I. Introduction.. . , . , . . , , . . . . . , . , . , . , . . , . . . , . , , . , . . . . , . . , . , . , . . . . , . . , . . . 11. Actual and Potential Functionality of Amino Acids. . . , . . , . . . . . , . . . , . 111. Classification of Mechanisms of Preferential and Selective Cleavages.. . . . A. Mechanisms of Preferential Cleavage through Neighboring Hydroxyl Groups.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mechanisms of Preferential Cleavage through Neighboring Carboxyl Groups.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . C. Preferential Cleavage through Formation of Dehydropeptides . . . . . . . . D. The Tertiary Amide Group. . . . . , . . . . . . . . . . , . , . . . . . . . . . . . . . . . . . . . . . . . IV. Principles of Selective Cleavage.. . . . . . . . . . . . . . , . . , . . . . . . . . . . . . . . . . . . . . . . A. Intramolecular Participation of Amide Groups in Pept,ides of Unsaturated Amino Acids. . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . B. Selective Cleavage of Tryptophan Peptides. . . . . . . . . . . C. Selective Cleavage of Tyrosine Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Selective Cleavage of Methionine Peptides.. . . . . . . . . . . . . . . . . . . . . . . . . . E. Cleavage of Histidine Peptides. . . . , . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . F. Cleavage of Simple 7-Aminobutyryl and 7-Glutamyl Peptides . . . . . . . . G. Attempted Cleavage of 0-Tosylhydroxyproline Peptides. . . . . . . . . . . . . . V. Selected Applications. . . . . , . . ... .. ... .. .. , . ., .. .. .. ... ... ... . ... . . . . , .. ... .. . ..... . ... .. . .. .. ... . . .. . . . . . A. Tryptophan Titration. . . . B. Selective Cleavage of Tryptophyl Peptide Bonds in Serum Albumins. . . C. “Buried” and “Exposed” Tryptophan in Tobacco Mosaic Virus (TMV) Protein . . _ . . . . . . . . _ ., . . , . . , . , . . _ _ _. _ _. _ .. . .. . . _ . . . . . . . . . . . . . . . . . . D. Cleavage of a n Antibiotic Cyclopeptide, Gramicidin A. . . . . . . . . . . . . . . E. Selective Cleavage of the Six Tryosine Peptide Bonds in Ribonuclease. F. Selective Cleavage of the Four Methionyl Peptide Bonds in Ribonucle. . .. . . . . . ase and the Separation of the Fragments., , . . . . . . . . . . . G. Cleavage of the His-Thr Bond in the Hemopeptide of Mam .. , . . . . . . . . , . . . . . . . . . . chrome C with N-bromosuccinimide.. . . . H . Selective Modification of Eneymogen-Enzyme Pairs. . . . . . . . . . . . . . . . . . VI. Induction or Prevention of Enzymatic Cleavage by Chemical Modification. VII. Conclusion.. , . , . , . . . , , . . . . . . . , , . . . . , . , . . . . . , . . , . , . . . . . . . . . . . . . . . . . . . . . . References.. . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . .
225 225 229 232 235 237 239 252 265 270 273 275 277 277 284 285 289 291 294 297 298 310 313 314
I. INTRODUCTION The late K. LinderstrGm-Lang is to be credited with the introduction of tlhe descriptive terms “primary,” “secondary,” and “tertiary structure” 221
222
B. WITKOP
which have made possible a better classification of the architectural elements of a complex protein molecule (Linderstrflm-Lang, 1952). During the last>decade the primary structures of more than a dozen important peptide hormones and larger protein representatives have been largely clarified and, in part, confirmed by synthesis (Table I). The methodology which is used in the determination of primary sequences of amino acids in proteins, reviewed recently by Thompson (1960a), has now become a routine and standard procedure. The matching of overlapping sequences of a multitude of peptides, obtained by enzymatic degradation, has become almost an accounting procedure. However, several recent statements underline the acute shortage of better methods for peptide cleavage : “Many proteins contain large parts of the molecule (cores) that are not attacked by trypsin and chymotrypsin and hence are likely to be difficult to study. Pepsin, papain, and elastase are three enzymes that can probably be used but they do not show such great specificity as trypsin and chymotrypsin” (Sanger, 1961). “Where peptide chemistry can make a contribution toward the proof of protein structure is in the application and continued formulation of degradative techniques. Since fragmentation of the protein molecule to smaller peptides is probably the key reaction in degradative processes, the search for specific reagents for the selective cleavage of various peptide bonds and the standardization of existing techniques is of prime importance” (Katsoyannis, 1961). The selectivity of an enzyme as specific as trypsin is no longer adequate when proteins with more than 100 amino acids are involved. In such a case it would be convenient to have a cleaving agent that splits just one or two peptide bonds selectively, possibly next to an amino acid that occurs only once or twice in the protein molecule. So far no enzymes are known that would perform such a task. However, very recently a number of nonenzymatic methods have been discovered which cleave peptide bonds next to amino acids such as tryptophan, tyrosine, histidine, or methionine (Witkop, 1960, 1961). Some of these chemical cleavages are highly selective and occur in places unattackable by enzymes. These new selective agents promise to be of use in the following respects: (a) selective fragmentation of large protein molecules into a very limited number of major subunits; (b) independent “auditing” of primary sequences arrived at by the “accounting” method; ( c ) rapid determination of sequence variations next to certain amino acids as a function of genetic mutations or species differences; (d) determination of the influence of secondary and tertiary structure on the reactivity of certain functional groups or reactive sites; ( e ) correlation of enzymatic or hormonal activity with progressive selective modification of certain reactive centers; (f) preparation of active fragments from larger enzymes by pinpointed, limited chemical cleavage.
TABLEI Survey of Peptides and Proteins Whose Primary Sequence, Partial, or Total Synthesis Has Been Completed and Which Have i n Part Been “Audited” by Nonenzymatic Methods Compound
Number
of amino
acids
Oxytocin
8
Vasopressin
8
Melanocyte-stimulating hormone a-MSH (from horse pituitaries) 8-MSH
13
Primary sequence du Vigneaud et a l . (1953b) ; Tuppy (1953); Tuppy and Michl (1953) du Vigneaud et al. (1953a); Acher and Chauvet (1953)
Glucagon
29
Dixon and Li (1960); Harris and Lerner (1957) et al. Geschwind (1957) Bromer et al. (1957)
ACTH
39
Davis and Bell (1955)
Insulin
51
Ryle el al. (1955)
Ribonuclease
124
Hirs et al. (1960)
Hemogloblin A a-chain &chain
141
TMV protein
158
al. Braunitzer el (1960a, 1961b) Braunitzer et al. (1960b, 1961b) Anderer et al. (1960a,b); Tsugita el al. (1960)
Papain
185
Papain active fragment
18
146
6045
Light el al. (1960) (incomplete) Hill and Smith (1960) 223
Synthesis and “auditing” du Vigneaud (1956)
et al.
Bodanszky et al. (1960a)
Hofmann et al. (1960); Harris and Lerner (1957) Schwyzer et al. (1959) Audited next to Try; Patchornik et al. (1958a, 1960) Fully active 23-peptide; Hofmann et al. (1961) Partial regeneration of activity from inactive A and B chain; Dixon and Wardlaw (1960) Reductive stretching and oxidative reactivation, White (1960). Audited next t o Met; Gross and Witkop (1961) and T y r ; Cohen and Wilson (1961) Audited next t o Met; Braunitzer (1961) Audited next to Try; Ramachandran and Witkop (1959) Total synthesis of active enzyme fragment possible
224
B . WITKOP
This chapter is a preview rather than a review. Most of the selcctive chemical methods for peptide cleavage have been thought about or tested only very recently. Although these methods have been applied to simple peptide models, only a few field studies with enzymes and proteins have been pursued. However, these chemical methods introduce a new challenging approach which may make possible structural work on more complex proteins with large molecular weights. TABLE I1 Amino Acids Possessing Additional Actual or Polenlial l’unctianal Groups Amino acid Cysteine Cystine Methionine Serine, threonine (8-hydroxyprolinea) yHydroxyproline (r-hydroxyarginineb.~) H ydroxyl ysined Aspartic acid, asparagine Glutamic acid, glutamine Lysine Arginine Tyrosine Tryptophan Histidine
Reactive site 8-Sulfhydryl 8 ,b’-Disulfide 7-Methylmercapto p-Hydroxyl 7-Hydroxyl 6-Hydroxyl p-Carboxyl or carbamyl 7-CarboxyI or carbamyl e-Amino Q-Guanido p-Hydroxyphenyl 8-Substituted indole 4 (5)-Substituted imidaaole
An unidentified imino acid from collagen (Ogle el at., 1961) has recently been identified as 8-hydroxyproline, presumably the trans-isomer (Irreverre et al., 1961). b erythro-7-Hydroxy-L-arginine has so far been observed only in Polycheira rufescens (cf. Fujita, 1960). 0 For a survey of other hydroxyamino acids see Greenstein and Winita (1961). d 6-Hydroxylysine occurs not only as a regular building stone in collagen but alvo in enzymes such as trypsin and chymotrypsin (Viswanatha and Irreverre, 1960). 0
11. ACTUALAND POTENTIAL FUNCTIONALITY OF AMINOACIDS Table I1 summarizes those amino acids that contain more than an unreactive aliphatic chain, namely a reactive site which may be a functional group in the traditional sense such as sulfhydryl, thiomethyl, hydroxyl, carboxyl, carbamido, amino, or guanido, or may be an activated aromatic ring or heterocycle such as the phenolic part of tyrosine, the pyrrole unit in troptophan, and the imidazole part in histidine. Phenylalanine would only be considered in this connection as the reactive di- or tetrahydro derivative ring. N-Peptides derived from proline and hydroxyproline are in :L separate class because they are tertiary amides carrying no proton :it the nitrogen atom. I t may be possible to utilize this special feature for a prefereiitial cleavage under propcr conditions.
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
225
111. CLASSIFICATION OF MECHANISMS OF PREFERENTIAL AND SELECTIVE CLEAVAGES Table 111 summarizes published and unpublished information on the nonenzymatic cleavage of peptide bonds (cf. Witkop, 1960; Cohen and Witkop, 1961; Witkop, 1961). At this point it becomes useful t o make a clear distinction between preferential (competitive or hydrolytic) and selective (noncompetitive, nonhydrolytic) cleavages, which are governed by different principles and mechanisms.
A . Mechanisms of Preferential Cleavage through Neighboring Hydroxyl Groups Preferential cleavage in the classic sense is observed under conditions of partial acid or, less frequently basic hydrolysis. Emil Fischer demonstrated the partial hydrolysis of silk fibroin by the action of strong sulfuric acid or concentrated hydrochloric acid at temperatures varying from 16” to 36°C for up to several days (Fischer and Abderhalden, 1907). These classic studies on various fibroins of different origins, which extended over a quarter of a century, (cf. Abderhalden and Brockmann, 1930) , established the lability of N-acetylserine peptides toward dilute acid, an observation which was confirmed later by much more refined studies using chromatographic (cf. Synge, 1943) and tracer techniques (Levy and Slobodian, 1952; Slobodian and Levy, 1953). The historic developments on the partial hydrolysis of proteins have recently been authoritatively reviewed in detail (Greenstein and Winitz, 1961). The labilizing influence of a neighboring hydroxyl group on the acid hydrolysis of serine or threonine peptides (I) is due to the acid-catalyzed breakdown (IV) of the cyclic hydroxyoxazolidine tautomer (11, 111) which is probably present in terms of a microscopic equilibrium (cf. Cohen and Witkop, 1961). A different O+ /R HO “NH I
I (1)
HOxC/R
. 0’
’ (n) /
-c-cI
H-&b/R
‘ y ----IHa-
/g
co.
0 ’ z$\
-Ho-c-cI
(In)
A
- 1
1
0 ’
ys
-7-7
(IV)
mechanism underlies the acceleration of the C-amide bond hydrolysis in hydroxy acids of which a quantitative study has been made recently (Zurn, 1960). Figure 1 and Table IV show the relative rates of hydrolysis under mild acid conditions. The slight increase in rate of hydrolysis of glycolic acid amide versus acetamide is probably due entirely t o an increased inductive effect. I n the amides with hydroxyl groups in the y-, 6-, or (most surprisingly !) a-positions, direct interaction of hydroxyl with the amide carbonyl (VI) leads t o marked acceleration of hydrolysis via amino-
TABLEI11 Surve.q in the Preferential or Selective Cleavage of Peptide Bonds . of -Principles and Reactions Emploued . Amino acid
Serine, threonine
Functional group
8-Hydroxyl DFP-Serine
Aspartic (Asparagine)
j3-Carboxyl
Glutamic (Glutamine) Tyrosine
7-Carboxyl
Tryptophan
8
0,
Histidine
Strong acids (H2SO4 H2F2, etc.) 1. DFP 2. Hz0, 100°C HOAC,pH 2-3,115'C or 0.03 N HCl, 105'c HCI in HOAc NBS", NBAb
7,b-Double bond of phenol 7 ,&Double bond of indole
NBS, NBA, HIOP, etc.
7, &Double bond
NBS, NBA
&!+Methyl
Cysteine (Cyst_.ie) 8-Mercapto
Hydroxyproline
Tertiary amide
Na in NHI
7-hydroxyl
solvolytic agents
b
0 Acyl shift
Special &elimination Preferential hydrolysis Preferential (?) hydrolysis Selective displacement Selective displacement displace -
Selective displacement 1. CH3Br in HOAc Selective 8-elimination and pref. hyor FDNB a t pH 6 drolysis 2. H20, 100°C, 2 hr. &Lactone formation Lactonizing agents Preferential amide Na in NHa cleavage
I-Hydroxyl Tertiary amide
N-Bromosuccinimide.
---t
ICHtCONH2, BrCN
I-Hydroxylysine Proline
a
N
Selective ment
of imidazole
Methionine
N-Bromoacetamide.
c
Yield
Type of cleavage
Cleaving agent
Preferential amide cleavage Displacement of 0-Ti r-lactone formation
Cf.Table VII, p. 252.
Reference
60% (impractical for many proteins 30-80%
Elliott, 1953; Narita, 1959; Fasman, 1960, Iwai, 1960 Patchornik et al., 1961
4545%
Yaron et al., 1961; Schultz et al., 1961
7040%
Vajda, 1959
So-so% (models) 20-3070 (proteins) 6040% (models) lW%(proteins) 1535%
6040% (models and proteins) *go% Not tested So far not tested
ichmir et al., 1959; Schmir and Cohen, 1961 Patchornik et al., 1958,1960; Ramachandran and Witkop, 1959 Shaltiel and Patchornik, 1961; Cohen and Schmir, 1961; Cohen and Wilson, 1961 Lawson et al., 1961; Gross and Witkop, 1961 Patchornik et al., 1961 Zahn and Traumann, 1954 Ziirn, 1960 Hofmann (1960a), (Cleavage in special model compounds) So far not tested
So far not possible
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
227
lactol (VII) and lactone. In the initial step the amide adds (V) a proton (VI). Recent nuclear magnetic resonance data are consistent with 0protonation of amides (Fraenkel and Niemann, 1958; Katritzky and
,r-Hydroxycapronarnidr 8-Hydroxywlerornide
B
7-Hydraxybutyrarnido
v)
35
2P
:O0
800 800
400
I200
MIN.
FIG.1. Hydrolysis of hydroxy acid amides in 1.0 N HCl at 30°C and concentration of 0.1 M. From Ziirn (1960). TABLEIV Rate Constants for the Hydrolysis of Hydroxy Acid Amidesasb Amide
k [min-'] X 1va
Amide
k [min-11 X
Acetamide Butyramide VaIeramide Capronamide
0.56 0.37 0.37 0.37
Glycolic acid amide 7-Hydroxybutyramide 8-Hydroxyvaleramide e-Hydroxycapronamide
0.75 6.5 16.8 Very fast
10-3
Zurn, 1960. In 1.0 N HCI at 30°C at c = 0.1 M .
Jones, 1961); in strongly acidic solution the oxygen of an amide is evidently more basic than nitrogen. R
(V)
R
( VI)
R
(VII)
The assistance from a hydroxyl group in the hydrolysis of an amide bond either via the (largely reversible) hydroxyoxazolidine or the (largely irreversible) lactone mechanism leads to preferential cleavage of peptide bonds next to serine and threonine, on the one hand, and to lactones of
228
B. WITKOP
hydroxylysinc and homoserine, on the other hand. With regard to the latter pair only a few observations hstve been recorded. Vor example, allohydroxylysylglycinamide (VIII) liberates glycinamide (XI) under mildly acidic (IX) conditions (Zahn and Zurn, 1956). AH- 2 kcal
CHz--NH3
HzN-CH2-
CONH
(XI)
(XI
Homoscrinc lactone (XII) easily forms the diketopiperazine (XIII) which is spparcntly in equilibrium with the tricyclic addition product (XIV) to judge from its chemical transformations and the relative ease of hydrolysis (Fischer, 1907; Snyder, 1942). Even the lower thio analog, thc diketopipcrazine of cysteine (XV) opens with remarkable ease to give the hydrochloride of cysteinylcysteirie (Greenstein, 1937). CH,-
OH
I
HO- CH,
(rn)
(XI)
&q+
0
HC1
(XIV)
cys-cys
METHODS FOR CLEAVAGE AND MODIFICATION
OF PROTEINS
229
The preferential cleavage of peptide bonds next to 6-hydroxylysine in collagen by the controlled action of acid in the cold has yet to be studied. In the bicyclic peptide phalloidine where there are three eligible hydroxyl groups, one p-hydroxyl in a threonine residue, and a y- and 6-hydroxyl group in the y ,&dihydroxy-L-leucineresidue, the controlled action of sulfuric acid leads to formation of the y-lactone only with selective cleavage of one peptide bond (Wieland and Schopf, 1959). HOCH, OH C ‘’
H,C
H ? C H - - ~ - - CO- CH -m--co--c’ \ I I HCOH NlC/
/ \
CH,-
/ o=y.-- C L X C H 2 HN I
H3C-HC
ocI
H
I
HN-CH-CO I HCOH
I
HZ
co
‘ S --CH,
//CO-NH-C-CH,I
I
-NH-CH
H
I
CHJ
Phalloidine 0.2 N H,SO, lOO”C, 30 min
H2
c, \ /
H3C
acH2 1
H,P
CH--MI-CO-CH-NH-CO-C
A0’
o\ Hsy H3C-HC
I
I
Lo
HOCH,
H
S
I CO-NH-CH-CO-NH I HCOH-CH,
-CH, I
HC-Cl O
\
HCOH
co I
N-C-CH, H $
”scco-Phalloidine- y-lactone”
R. Mechanisms of Preferential Cleavage through Neighboring Carboxyl Groups The preferential release of aspartic acid from proteins by dilute acid has been known for some time (Partridge and Davis, 1950). The difficulty is to find conditions under which the accelerating effect (Leach, 1955) of the 8-carboxyl group on the cleavage of the aspartyl peptide bond surpasses the preferential hydrolysis of peptides next to serine, or threonine. The early attempts to utilize this method in the hydrolysis of the A chain of oxidized insulin led to incomplete release of aspartic acid and t o a large variety of peptides liberated by cleavage of peptide bonds next to serine, threonine, and cysteic acid (Thompson, 1960a; Naughton et al., 1960).
230
B. WITKOP
Recently t w o new modifications have been introduced for the preferential release of frre aspartic acid. The methods of Schultz et al., (1957); Schultz and Delavslii (1058); Schultz et aE. (1961), and Yaroii et aE. (1961) USC 0.03 N HCl and 4.5 7'0 acetic acid, respectively, and make use of the older observation that the preferential hydrolysis is a function of the p H and not of the nature of the acid concerned (Blackburn and Lee, 1954). H2C-C
I?
H,C-C
/P
HC-C-NHR HNFC -R'
HN- C-R'
(XW
H2C-C
118
B 0
HzN
(XVII)
R'COOH
"T ' -;O
H2O
P"
HC-C-NHR
I
H2N C,,-R' 0
(XVIII)
RM,
H2C-COOH
I
HC-COOH HzN R'COOH
+ R'CooH
HZN
Any mechanism written for this preferential cleavage must take into account t,he double cleavage on both sides of the aspartyl residue that leads eventually to liberation of free aspartic acid (XXI). The nucleophilic concerted interactions (XVI-XVIII) pictured in the proposed mechanism may lead t o a bicyclic orthoesteramide (XVII) which would collapse with the release of aspartic acid (XXI). Although the details of this tentative mechanism remain to be established, there is ample precedent for the formation of anhydride intermediates (XX) in intramolecular electro-
METHODS FOR CLEAVAGE AND MODIFICATION
231
OF PROTEINS
philic-nucleophilic catalysis (cf. Garrett, 1960; Bender, 1960; Bruylants and Kezdy, 1961). Table V summarizes experiments on the preferential cleavage of aspartyl peptide bonds by the action of 4.5% acetic acid. Such a met'hod is desirable for the rapid assay of species differences in simple peptide hormones, such as the melanocyte-stimulating hormones (p-MSH) of porcine, bovine, or human origin. There the species difference is located after the TABLEV Preferential Cleavage of Aspartyl Peptides b y the Action of 4.6% Aqueous Acetic Acid* ~
Compound Poly -L-aspartic Copoly -L-aspartyl-L-lysine.HBr Ribonuclease Asparagine Aspart ylgl ycineamide Multi-poly-L-prolyl-~-benayl-oL-aspartylpoly -oL-lysine
pH
3.2 2.0 3.2 2.7 2.9 2.6
'rtFy' TLy 115-120 100 100 100 100 100
24 24 24 48 48 48
aspartic acid released 80 87 46 94 47 G3
% ' of Free amino acid released
Control experiments DL-Leucylglycine L- Alanylgl ycine Alanylalanine Copoly-L-glutamyl-L-lysine.HBr
of Free
3.0 3.1 3 .O 2.0
100 100 100 100
48 48 48 48
3 Clycinc G Clycine 5 Alanine -1,
Yaron et al., 1961. The material was insufficiently soluble, therefore t h e amount of cleavage could not be determined. In t h e supernatant of the reaction mixture an insignificant amount of glutamic acid was detected. 0
Ir
first aspartyl residue (cf. Hofmann, 1960 a, b). The method, however, has all the disadvantages of preferential cleavage as shown by the significant random cleavage in the control experiments. Schultz observed (Schultz et al., 1961) that on partial hydrolysis of a commercial preparation of ribonuclease with 0.03 N HC1 for times varying up to 48 hr a t 105"C, up to 14 out of the 15 bound aspartic residues were liberated. He recommends the method for rapid comparative "fingerprinting" of the resulting peptides from proteins such as human and animal sera, heme proteins, pepsin, and albumins. I n addition t o cleavage of aspartyl and asparaginyl peptide bonds, the rupture of glutamyl and glutaminyl linkages has been shown to occur on a
232
n.
WITKOP WITKOP
limited scale and with moderate preference on treatment of model peptides and insulin with hydrogen chloride under anhydrous conditions (Vajda, 1959a, b).
C . Preferential Cleavage through Formation of Dehydropeptides This approach, under study a t the Weizmann Institute (Patchornik et al., 1961), proceeds in three stages. At first the p-hydroxyl of a serine or threo-
-R‘Xo
nine (XXII, X = 0),or the @-mercaptogroup of cysteine (XXII, X = S), is converted into a derivative (XXIII), OR’ or SR’, that will make a good leaving group, R’OQ or R’Se, in the subsequent p-elimination (XXIII) . The second step, the @-elimination,is brought about by a mild base such as bicarbonate. The resulting compound (XXIV) is a dehydropeptide. Such dehydropeptides are known to undergo easy hydrolysis (Bergmann and Grafe, 1930; Clarke and Inouye, 1930) under mildly basic or acidic conditions. In the third step mild hydrolysis leads to the formation of a CONHZ-terminal (XXVI) and an amide of pyruvic acid (XXV). In this sequence the initial acylation or alkylation of P-OH or P-SH, respectively, as well as the p-elimination step are largely selective under proper conditions. The breakdown of the resulting dehydropeptide in the third step is a preferential hydrolysis, although the conditions have been modified to advantage by heating the dehydropeptide for several hours in a sealed tube at 100°C. This heating process restricts the method to sequence studies and makes it unsuitable for selective cleavage and modification of sensitive proteins without concomitant denaturation. This new approach to peptide cleavage may be amenable to further refinement and eventually may lead to selective cleavage at room temperature.
METHODS FOR CLEAVAGE AND MODIFICATION
OF PROTEINS
233
The leaving group in this procedure may be modified so that only cysteine peptide bonds are cleaved. Unlike amino or hydroxyl groups, mercapto groups react with fluorodinitrobenzene (FDNB) at essentially neutral pH. The formation of the dinitrophenylcysteine (DNPS) derivative (XXVIII) (Zahn and Traumann, 1954) is rapid and can be followed in a pH-stat. With reduced glutathione the reaction was completed within a minute. When the isolated DNPS-glutathione was dissolved in 0.2 N NaOH the &elimination (XXVIII, XXIX) was complete within a half-hour. The difference of the ultraviolet spectra between the original DNPS-derivative and the liberated dinitrothiophenolate anion (XXX) permits an easy spectrophotometric assay of the p-elimination which proceeds in about 90-100 % yield. NH-OC -HN
-c
I
NH -
NH I
I
I
co
yo
- OC-HN-C--H
--H
CH, I SH
-0C-HN-C
HOQ
IJ
FDNJpH 6-7
yo II
Cn,
NO,
NO*
Measured at h =408mp
Cysteine derivative
(xxvm)
(XXWI)
(XXX)
Complications in this procedure when applied to proteins might arise from the reaction of imidazole units with FDNB (cf. Gundlach et al., 1959a, b) or from the possibility of intramolecular assistance from a neighboring tlmide (imidole) group in the process of elimination, as is pictured for glutathione (XXXI) (Calvin, 1954). Such a reaction could lead to an oxazoline (XXXII).
4; bN02 O2N
(7
,,(,
\4
C,
+
_____._. ?
8 4 ! 2 m y 2
CH-C-NH
HOOC NH,' N ' H (XXXI)
0 /I
CH,COOH I
J K " ; H : " 'H
HOOC NH,
\N/
CH -C -NH- CH2- COoH
1
(XXxlI)
Cysteine peptides may be converted to sulfonium derivatives by reaction with alkyl halides. The resulting sulfonium salts undergo p-elimina-
234
B. WITKOP
tion. By the use of the water-soluble m-carboxy-o-xylylene dibromide (XXXIII) the second alkylation step to the sulfonium salt (XXXV) be-
r
B
-
z
H
c R-SH i f
-HBr
+
HOOC
R
-
-
f
n COOH
Br-H,
CH,-Br
L,
R-
I
*"'-n H,
I
Br e
C'
COOH
H,
(XXXV)
comes intramolecular (XXXIV). Both steps proceed under mild conditions and may be followed successively in a pH-stat. /%Elimination in phosphorylated hydroxyamino acids (XXXVI) has been observed, e.g., in the breakdown of the N-acyl-O-(di-O-phenylphos(R"O),POOCH,-CH-COOR I NH-C-R' II 0
(XXXW
-
(R"O),PO. OH + CH,=C-COOH I NHCOR'
(XXXVII)
(XXXVIII)
CH,-CO-COOH
1
+ H,N-C-R' I1
0
pho)scrine citer (XXXVI) t o diphenyl hydrogen phosphate and N-acylaminoacrylic acid (XXXVIII) (Riley et aZ., 1957) or in the base-catalyzed dcphosphorylntion of phosvitin which causes an increase in ultraviolet absorption indicative of the formation of dehydroalanyl residues (Mecham and Olcott , 1949). The selective phosphorylation of the active serine hydrosyl of enzymes such as cholinesterase, chymotrypsin, trypsin, etc., by diisopropylphosphorofluoridate (for reviews cf. Boyer, 1960; Perlmann, 19.55) leads to a modified enzyme containing a single diisopropylphosphate (DIP) ester bond. Preliminary observations point to the formation of 30-100 %, of pyruvic acid on treatment of, e.g., diisopropylphosphoryltrypsin in watcr at pH 7 at 100°C for 24-48 hr (Patchornik et d., 1961).
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
235
In this case the two steps, p-elimination and hydrolysis of the resulting dehydro enzyme were done simultaneously. This procedure may facilitate the task of establishing the chemical structure of the active site of esterases and peptidases (cf. Cohen et al., 1959; cf. TomS9ek et al., 1960; Sanger and Shaw 1960). Complications may arise here through base-catalyzed dismutations of pyruvoyl peptides, which are observed in the conversion of pyruvoylglycine into alanine by the action of base (Fu et al., 1952; cf. Wieland et al., 1958).
D. The Tertiary Amide Group The N-acyl groups in bound proline and hydroxyproline differ from all the other peptide groups in being fully substituted tertiary amides. Whereas secondary peptides (XXXIX) may form anions (XXXX) by abstractioii of a proton from the nitrogen atom, the formation of an anion (XXXXII) from a proline peptide (XXXXI) would have to proceed with cleavage of
-c--m-- eOH
-c-N-
b e
II 0
(XxXx)
(xxI(IX)
LyQI
o=c
I
(xxxxr)
m e c 0 11 -
o=c-x I
)-(
the amide bond. Preliminary observations (Francis and Witkop, unpublished) point to cleavage of certain N-acyl- (hydroxy)prolinamides on treatment with sodamide-sodium in liquid ammonia. These observations on simple model compounds have so far not been successful with regular peptides of hydroxyproline. However the principle of such a preferential sodamide-sodium cleavage may be worth further exploration. Special intramolecular assistance may increase this effect considerably. For example, in synthetic studies on a-MSH an €-N-tosyl group of the sequence Lys-Pro (XXXXIII) was removed by sodium in liquid ammonia resulting in cleavage of the Lys-Pro bond, XXXXIV + XXXXV XXXXVI, (Hofmann, 1960a, b). The proximity of the N-tosyl anion to the t,ertiary N-acyl group may facilitate cleavage by sodium in liquid ammonia. Similarly, as the result of an 0 -+ N acyl migration, the €-amino group of natural or erythro-6-hydroxylysine lactone (XXXXVII) participat,es in the
+
236
B. WITKOP
QCONHR 0 (XXXXVI) n c o N H l 3
-c\p
.7\ccp
QCONHR TsN
I
TsN-
C
I AH (CH,): “ H R ’
A H
(CH,C ‘NHR’
(XXXXIII)
--(
TsN-C
90
1 ,ANHR,
(CH,),
(XXXXIV)
(XXXXV)
formation of an t-lactam hydrochloride (XXXXVIII) (Zahn and Zurn, 1958). This interaction is stereospecific : N ,N‘-dicarbobenzyloxyallo(2hreo)d-hydroxylysine lactone (XXXXIX) on hydrogenolysis does not show this intramolecular acyl migration to an t-lactam.
R
=
H or Cbz
OH
(XXXXVII)
(XXXXVIII)
dlCbZ
CbZHN-CH, H
Hz, Pd
,
-
allohydroxylysine
(XXXXIX)
Activation of the carboxyl in ornithine (cf. Bell, 1956) and argiriiiie (Bodanszky and Sheehan, 1960; Zervas et al., 1961) derivatives leads t,o intramolecular formation of 6-lactams. Activation of the carboxyl in lysine derivatives leads to unwelcome side reactions due to interaction with the €-amino function (Harington and Moggridge, 1940; Cipera, 1961). A convenient model compound for the further study of this cleavage would be N-tosyl-y-aminobutyrylprolinamide.It would still have t o be established that the cleavage of such N-prolyl peptides is preferential by comparison with similar peptides of primary amino acids. Liquid ammonia alone is known to be a good solvent for proteins and enzymes with little effect on activity or secondary structure after recovery of the initial material (Ellenbogen, 1955).
METHODS FOE CLEAVAGE AND MODIFICATION OF PROTEINS
237
The disadvantageous feature of the preferential cleavage is its dependence on hydrolytic conditions. In the final analysis, the method relies on acceleration of hydrolysis by intramolecular assistance from functional groups or on inherent instability as is the case with dehydropeptides. While the now classic elucidation of the primary sequence of insulin made much use of acid hydrolysis, more recently acid-catalyzed inversions of dipeptide sequences (Sanger and Thompson, 1952; Tuppy and Bodo, 1954; Schaffer et al., 1955) have been observed and have led to a trend away from acids and back to enzymes. Cleavage by preferential hydrolysis in a number of special cases has been of practical interest for the study of primary sequences. For instance, the N ,0-acyl shift in clupeine with concentrated sulfuric acid at 20°C for 4 days, followed by hydrolysis of the ester bonds by 6.0 N HC1 at 2loC, cleaved peptide bonds next to serine and threonine t o the extent of 50-70% and 10-20%, respectively. Nonspecific fission of other peptide bonds was limited to 0.5-1.0% of the arginine N-linkages present (Iwai, 1959, 1960). Deamination with nitrous acid, or acetylation of the amine groups of the rearranged clupeine raised the cleavage to 80-9070 of the tot,al serine bonds present (Iwai, 1961). Although in this case the fission has been termed “selective” this term should better be reserved to iiorihydrolytic methods of specific cleavage as described in the following.
I V . Principles of Selectioe Clcavagr: Selective cleavage still makes usc of intramolecular assistance, hut departs from conventional nucleophilic hydrolytic agents in favor of electrophilic agents or groups. The roles in this case have been reversed. In hydrolysis the amide carbonyl (L) serves as attractant (LI) for nucleophilic groups; in the selective cleavage the negative end of the carbonyl
(L) ’
(LII) (displace-
ment)
-COOH + NH,-
0
(LIV)
( LIII)
dipole (LII), or the imidole tautomer of the amide, acts as an intramolecu-
238
B. WITKOP
lar nucleophilic agent, attacking a y- or &carbon (LIII) atom from which electrons or leaving groups can be displaced (LIV). The participation of amide bonds in displacement reactions has been demonstrated by Winstein and his school (cf. Goodman and Winstein, 1957). In the case of 3-benzamidopropene (LV) the yields of the oxazoline L(V1) are highest when the bromine addition is carried out in weakly nucleophilic solvents
such as acetic acid. Side reactions leading to dibromides and bromo ethers are observed in methanol and other nucleophilic solvents. Optimal intramolecular interaction is observed when the resulting iminolactones are five-membered. This means that the displacement process should occur a t the y-carbon atom with respect to the carboxyl (carbamyl) group. In the simplest case, y-bromobutyramide (LVII) will tautomerize to
BPr Q N W R A
H
- Q;:R & Stability (LWI)
(LVII)
Bp rm 0 fusion
-
Do
Cl3HIl n-W1 C,HS Benzyl
+
-
+
I
I R
R
( LIX)
( LX)
iminotetrahydrofuranhydrobromide (LVIII) on heating to 100°C (Stirling, 1960) or by ethanolysis, with formation of y-ethoxybutyramide as a side product. On the other hand, intramolecular displacement of the y-bromo atom may be brought about by the amide anion (LIX) under strongly alkaline conditions. In this case a pyrrolidone (LX) is formed. Neither condition, pyrolysis nor alkali fusion, is acceptable for proteins. The criteria for a useful selective peptide cleavage, applicable to the study of sensitive enzymes without causing side effects or denaturation, are: ( a ) the reagent must react with only one reactive site; (b) the rate of reaction should be rapid; ( c ) addition of the reagent to the reactive site should yield a highly unstable intermediate; (d) the great instability
METHODS FOR CLEAVAGE AND MODIFICATION
OF PROTEINS
239
of this intermediate should make for a rapid, concerted 1,5-intramolecular displacement reaction by only one peptide group, namely, the C-peptide group; ( e ) the resulting iminolactone should break down immediately into a C-terminal lactone fragment and a new NHs-terminal; df) the entire sequence of reactions must occur rapidly in aqueous buffer solutions a t neutral or slightly acidic p H and at room temperature. The criteria, admittedly, are stringent and read more like a code of ethics for which a new group of enzymes rather than some well-known chemical reagents would qualify. However, the following examples may illustrate that these conditions are indeed fulfilled by a number of simple oxidants.
A . Intramolecular Participation of Amide Groups in Peptides of Unsaturated Amino Acids Amides and peptides (LXI) of N-acylated DL-allylglycine (m-%amino4-pentenoic acid) (Albertson, 1946) or DL-methallylglycine (DL-2-amino-4-
methyl-4-pentenoic acid) (Goering et al., 1948) react in aqueous buffer systems or water with N-bromosuccinimide (NBS) (LXII, LXIII) under liberation of ammonia or the peptide component (LXIV). Figures 2, 3, and 4 show that the liberation of ammonia, glycine, or glycinamide is a linear function of the added reagent and that after the addition of 1-2 moles of NBS 60-80% of the amine component is liberated, as measured by ninhydrin assay. On a preparative scale the primary amides gave products different from the lactones obtained from the allylglycine peptides. They were originally thought to be iminolactone hydrobromides (Craig, 1952), however, their
240
B. WITKOP
FIG. 2. The liberation of ammonia by cleavage of N-tosylullylglycinamide (A) and N-benzoylmethallylglycinamide (€3) ILR I I fuiiction of t,lie nmoririt o f NRS and of t,he pH. From Izumiya el al. (1962).
- A 8o
-
0
B WATER
1
2
0
I
2
MOLES NBS
FIG.3. The liberation of glycine from N-benzoylallylglycylglycine (A) and from N-tosylmethallylglycylglycine (B) as a funct8ion of NBS addition and of pH. From Izumiya et al. (1962).
insolubility in water, solubility in nonpolar solvents, analytical data, and content of one covalent and one active positive bromine led to their formulation as N-bromoiminolactones, a type of compound described earlier for imino ethers (Stieglitz, 1896). The initially formed iminolactone
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
241
MOLES NBS
FICA 4. The liberation of glycinamide from N-benzoylallylglycylglycinamideas a function of NBS addition and of pH. From Izumiya et al. (1962).
reacts with excess NBS to yield the water-insoluble N ,C-dibromoiminolactone (LXVI) . The allylglycylglycine peptide forms a soluble iminolactone which has no replaceable N-proton and breaks down to yield a
-\\\i , : - I 0"" - NH,-CH,COOH
R = CH,COOH
(LXVI)
HP
-HOBr
0
OH
- (R' = H)
H
(LXVIn)
( LXVII)
homogeneous lactone (LXVII) which has been assigned the cis-configuration (both hydrogens on the same side of the five-membered lactone ring) because of its easy conversion to allohydroxyproline (LXVIII) (Izumiya arid Witkop, 1962). This stereospecificity may be a result of an intra-
242
B. WITKOP
molecular transfer of positive bromine (LXV, arrows a) from an intermediate N-bromoamide formed initially by an exchange reaction with NBS. The bromonium or bromocarbonium intermediate then invites participation by the peptide group (LXV, arrows 6 ) . For steric reasons a fully concerted mechanism is not possible. A y,b-unsaturation in a six-membered amino acid such as the easily accessible amide of DL-baikiain (LXIX) (Burgstahler and Aiman, 1960) invites participation of the amide in the reaction with NBS to yield the bicyclic bromolactone (LXXI) of fixed configuration via the water-in-
qp; f j L fl
0
Br
Ts (LXIX)
Br (L x x )
Ts
Ts
(LXXI)
soluble N , C-dibromoiminolactone (LXX). No participation of the y ,aunsaturation and no liberation of glycine are observed when N-acylbaikiainylglycine is allowed to react with NBS. The participation of a y ,&double bond in a six-membered carbocyclic ring poses no problem even if it leads to a spiroiminolactone intermediate. Palustrine (LXXII), a representative of the interesting class of spermidinc alkaloids (Eugster, 1960) yielded a y-lactone (LXXIV) (vmaX 1770 cm-I) with N-bromosuccinimide, probably via the intermediate spiroiminolactone (LXXIII) and hydrolysis (Eugster, 1961). A similar XBS-cleavage has been observed by Patchornik (personal communication) with peptides of phenylalanine after reduction with Li in CH,KH,. 0 Br
( LXXII)
Br
Br
( LXXIII)
( LXXIV)
A further rcst,rictioii is imposed upon an isolated P,-y-double boiid in a
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
243
five-membered ring. N -Carbobenzyloxy-3,4-dehydro-~~-prolinamide (LXXV) does not react with NBS in acetate buffer a t p H 4 but only in phosphate buffer, pH 6, to yield, via a Hofmannamidedegradation (LXXVI, LXXVII), allylic bromination (LXXVII) and hydrolytic removal of bromo and amine function, an unusual crystalline dicarbinolamide (LXXVIII), formally the condensation product between maleic dialdehyde (cf. Hufford et al., 1952), and an amide. Compound (LXXVIII) still contains the original double bond as ascertained by nuclear magnetic resonance (NMR) studies and oxidation to N-carbobenzyloxymaleamic acid (LXXX). The ring structure is proven by oxidation, hydrogenation, and hydrogenolysis to succinimide (LXXIX) (Robertson, et al., 1961, Robertson and Witkop, 1960).
<-- *k=yo;:pkd-2;z CONH,
N
I
H
Br
N
Cbz I
Cbz
(LXXVI)
(Lxxv)
H
Br
N
(!!bz
(LXXvn)
H t Cbz
(Lxxvm)
b !tz ( LxxrX)
(Lxxx)
Succinamic acid ( LXXXI)
The participation of an isolated double bond in a five-membered ring in displacement reactions is apparently difficult or impossible, but fortunately becomes very easy when the double bond is conjugated with a n imino group and in the y ,&position relative t o a carboxamido group, as in tryptophan peptides.
244
B . WITKOP
B. SelectivP Cleavage o f Tryptophan Peptides I Ka11 ~ indole derivative electrophilic reagents may substitute or oxidize any one out of five or more clectronegative reactive sites. The introduction into the &position of the side chains with functional groups ftirthw increases the complexity of this picture. Indoleacetic acid (LXXXII), the plant growth hormone auxin, may undergo various transformations with NBS depending on the medium. In acetic acid substitution, hydrolysis and oxidation presumably lead to a hitherto unisolated dioxindole intermediate (LXXXIII) which, on hydrogenolysis of the nuclear bromine and the benzylic hydroxyl, yields oxindole-%acetic acid (LXXXIV) (Lawson and Witkop, 1961:t, b). This substance has recently been found in plants (Kliimbt, 1959) arid as a bacterial metabolite in the basidiomycete Hygrophorus conicus (Siehr, 1961). In tertiary butanol a labile p-hronio intcrmediatc (LXXXV) can be isolated (Hinman E t al., 1961) that may either lose HRr and, on ether cleavage, yield oxindole-%acetic acid
(LXXXV)
H,, Pd-C in HOAc
H
H
XM~X 253 (E, 25,500) 247 (c, 25,400)
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
245
(LXXXIV) or undergo further oxidation and base-catalyzed decarboxylation (LXXXVI) to the labile nonisolable p-methyleneoxindole (LXXXVIIa) , characterizable as the sodium bisulfate addition product (LXXXVIIb) . p-Methylene oxindole (LXXXVIIa) bears a spectral resemblance to the oxidation product of auxin produced by peroxidase or indoleacetic acid oxidase from the fungus Omphaliu $uvida (Ray and Thimann, 1956). Indolepropionic acid (LXXXVIII) (IPA) undergoes similar transformations. However, in this compound the oxygen of the carboxyl of the side
Pd, H,
in HOAc-HC1
(XC)
chain is in the favorable 1,5-position with regard to the active p-carbon of the indole system. Intramolecular participat)ion of this carboxyl in displacement reactions is to be expected. Indeed, a neutral oxidation product (LXXXIX) with the characteristics of a y-lactone is obtained from II’A with NBS (Lawson e2 al., 1960). It is easily hydrogenolyzed to the known oxindole-3-propionic acid (XC). The mechanism of this lactonization reaction is best pictured as a concerted intramolecular displacement of the carboxyl or carboxylate group on
246
B. WITKOP
the initially formed, highly labile bromonium intermediate (XCI) . Elimination of HBr (XCII) and further oxidation of the resulting indolenine lead to the crystalline spirodioxindole-ylactone (XCIII) . The same participation is shown by indole-3-propionyl peptides. I n this case, labile iminolactones (XCV ---f XCVI) are formed which hydrolyze easily to the lactone (XCVII) and the respective amino acids (Patchornik et al., 1960). When carbobenzyloxytryptophan was treated with successive increments of N-bromosuccinimide the initial indole spectrum (Fig. 5A, I), after the addition of 1.53 moles of NBS, changed t o an oxindole spectrum (Fig. 5A, 11) similar to that of the bromospirooxindole I1 (ezx7261 m p 10,300; € 3 ~ 3m p
1630). Figure 5B,I, 11, I11 presents these same changes expressed as difference spectra, the reference cell containing the starting material in the same concentration as the observation cell. I n the process of lactonization of L-tryptophan or its DNP-derivative a second asymmetric center is formed, probably stereoselectively, by induction from the existing center. Opening of this DNP-lactone and application of Hudson’s rule (cf. Witkop, 1956; Vanderhaeghe and Parmentier, 1961) has so far pointed to the D, structure for the spirooxindole-&carbon (Ramachandran, 196l), but this preliminary observation on a colored lactone derivative must await conformation from, e.g., a carbobenzyloxy lactone derivative. The correct steric representation by the Fischer convention of two asym-
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
I Cbz Try 11 Cbz Try + 1.53 moles N.B.S. A
I
Cbz Try
247
+ 0.6moles N.B.S.
I1 Cbz Try+ 1.2 moles N.B.S. IIICbz Try 1.5 moles N.B.S.
+ B
FIG.5. Ultraviolet spectra of Cbz-Try with N-bromosuccinimide in aqueous acetate buffer of pH 4.0. A. Recorded directly. B. Recorded as difference. From Patchornik et al. (1960).
metric centers, assumed to be L, and Dc , is shown in XCVIII or XCIX with the assumption that the phenyl group be considered a substituent on the Rosanoff C-atom; the N-ring methylene is part of the presumptive chain.
(XCVIII)
(XCIX)
These spirodioxindole lactones are opened by methanol, but not by ethanol, to yield methyl ester derivatives of dioxindole-3-propionic acid. This reaction proceeds even at room temperature in aqueous methanol. Refluxing anhydrous methanol converts the lactone of 5-bromodioxindole3-propionic acid (m.p. 200.5"C) to the ester (m.p. 118°C) within 1-2 hr. The use of methanol in the cleavage of water-insoluble peptides, such as
248
B. WITKOP
gramicidin A, should therefore be avoided. Aqueous ethanol has been used to advantage in such cases. The ability of tryptophyl peptide bonds to participate in the lactonization reaction was demonstrated, for example, with N-carbobenzyloxy-Ltryptophylglycine. Figure 6 shows that the decrease in optical density at 280 mp and the liberation of glycine proceed in parallel and reach a maxiI:
I
I
I
I
4
1
2
3
4
5
MOLES OF N-BROMOSUCCIN I M IDE
FIG. 6. The liberation of glycine from N-benzoyltryptophylglycine (IV, -.indole-3-propionylglycine .-.-),(V, -X-X -X -), and carbobenzyloxytryptnphylglycine (111, -0-0-0.)as a function of the addition of N-bromosuccinimide to the solution of the peptides in acetate-formate buffer a t pH 4. The decrease i n optical density a t 280 mfi (. . . .) reaches 100% after the addition of 1.53 moles of NBS. 3
.
From Patchornik et nl. (1960).
mum after the addition of 1.5-2.5 moles of N-bromosuccinimide (Patchornik et al., 1960). In an alcoholic aqueous acetate buffer at pH 4,glycine was liberated in 39 % yield. The maximum yield of glycine was obtained between pH 3 and 5 . The carbobenxyloxy group in Cbz-Try-Gly apparently affects the cleavage adversely, since benzoyltryptophylglycine and indole-3-propionylglycine liberated glycine to the extent of 55 %. The extent of cleavage in acetate buffer (Fig. 6 ) , measured colorimetrically, when plotted versus the amounts of N-bromosuccinimide, passed through a maximum. After complete oxidation of tryptophan, excess N-bromosuccinimide degraded
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
249
glycine. I n order to avoid such secondary oxidations a protective agent had to be added. Formic acid is known to be rapidly oxidized by N-bromosuccinimide in aqueous solution to yield COZ , HBr, and succinimide (Barakat et al., 1955). Accordingly, the addition of formate to the buffer solution, used for the cleavage reaction, to some extent protected the liberated amino acid from further destruction (Fig. 6),even when large amounts of N-bromosuccinimide were used. A large excess of N-bromosuccinimide should, however, be avoided. In the cleavage of most model peptides maximum yields were obtained after the addition of 2-3 moles of oxidant (Table VI). Similar yields, 47-60 %, have been obtained for various other model peptides such as the N-indole-3-propionyl derivatives of glycine, alanine, leucine, phenylalanine, and proline. Variation of the length of the indole 0-side chain showed that maximal cleavage occurs when 1-5 interaction is possible: The yields for 1,5- 1 ,6-, and 1 ,Pinteraction decreased in the order 53 % (ethyl indole-3-propionylglycinate) , 17 % (indole-3-b~tyrylglycinate) ,and 3 % (ethyl indole-3-acetylglycinate) . The labile bromonium intermediate should provide much more driving force for the concerted intramolecular displacement than an alternate 0-bromooxindole (CI). This was tested by the action of NBS on ethyl carbobenzyloxy-~-2-hydroxytryptophylglycinate (C) which was cleaved in
much lower yield (13%) than ethyl carbobenzyloxy-L-tryptophylglycinate (39%). Thus, the former cannot be a major intermediate in the cleavage reaction. The possibility of an N-bromoindole intermediate was eliminated by the demonstration that N-bromosuccinimide reacts with N-methyl indole-3-propionylglyciiie.
(CII)
(CIII)
The participat,ion of secondary amino acids, e.g., of proline, and the intermediate formation of quaternary iminolactones (CIII) (Craig, 1952)
TABLE VI Yields of Liberated Amino Acids i n the Cleavage of Various Tryptophan Peptides Under FOUTDifferent Sets of Cnnditaons" Yield of liberated ninhydrin-positivecleavage products
(%) Peptide
Carbobenzyloxytryptophylglycine Benzoyltryptophylglycine Indole-3-propionylglycine Indole-3-propionyl-~-alanine Indole-3-propionyl-~-leucine Indole-3-propionyl-o, L-phenylalanine Indole-3-propionyl-~-proline Ethyl indole-3-acetylglycinate Ethyl indole-3-propionylglycinate Ethyl indole-3-butyrylglycinate Ethyl carbobenzyloxy-L-tryptophylglycinate Ethyl carbobenzyloxy-L-oxytryptophylglycinate Ethyl benzoyltryptophylglycinate N-Methylindole-3-propionylglycine Indole-3-propionyl-p-nitroaniline a
b
(Patchornik et al., 1960). No p-nitroaniline detected in the ultraviolet.
Cleavage product
G~Y GlY GlY Ala Leu Phe Pro Gly-OEt Gly-OEt Gly-OEt Gly-OEt Gly-OEt Gly-OEt GlY p-Nit roaniline
With NBS in With NBA in With NBsin With NBA in formate-acetateformate-acetate LioAc LiOAc buffer buffer (PH 4.0) (PH 4.0) (PH 4.0) (PH 4.0) 39 55 55 53
53 47 60 3 53 17
39 13 51 65 O*
36 47 69 65 56 49 52 2 55 10 28 22
44 73
67 65 60 75 67 70 72 0
62
7 53 15 59
87
63 84 91 90 90 71
80 0 71 10 53 57 53 98
m
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
251
poses no difficulties to judge from the smooth cleavage of indole-3-propionyl-L-proline (CII) . Substituents at the amide nitrogen with strong electron-attracting groups influence the amide $ imidole t,automerism and prevent intramolecular participation and elimination. Thus, the p-nitroanilide of indole-3-propionic acid was not cleaved by N-bromosuccinimide (Table VI). N-Bromoacetamide, which reacts much more slowly than N-bromosuccinimide with indoles, cleaves peptides of indole-3-propionic acid and acyltryptophans in yields comparable with those obtained with N-bromosuccinimide (Table VI). Slight variations fall within the limit of error ( f 5 %) of the analytical method. The half-life of a 0.0001 M solution of indole-3-propionic acid in the presence of 3 moles of N-bromoacetamide (NBA) is about 7 min at room temperature, while N-bromosuccinimide reacts instantaneously. This is at variance with the general statement in the literature (Barakat et aZ., 1955) that N-bromoacetamide is considerably more reactive than N-bromosuccinimide (cf. Schmidt et al., 1926; Buckles et al., 1957). In a medium of 8-10 M lithium acetate at pH 4, however, the extent of cleavage of the model peptides with both N-bromosuccinimide and N-bromoacetamide increased markedly (Table VI). This increase in yield is probably due to the greatly decreased activity of water in concentrated salt solutions (cf. Robinson and Stokes, 1955). Under such conditions opening of the bromonium intermediate by water competes poorly with intramolecular participation of the imidole group. Although the cleavage in 8-10 M lithium acetate gives maximal yields for small peptides, markedly decreased yields are observed under such conditions in the cleavage of tobacco mosaic virus protein, lysozyme, and other proteins as will be described in Section V. The probable explanation is that lithium acetate, like lithium bromide, brings about macromolecular transformations which may change the accessibility of certain reactive sites in the protein or the strength of intramolecular hydrogen bonds (cf. Harrington and Schellman, 1957). The ensuing steric restraints may make the 1 ,&interaction of the tryptophyl side chains difficult or impossible. Steric inhibition of 1,5-interaction, as reflected by low cleavage yields, is especially noticeable in cyclic peptides, such as tyrocidin B (Gross et al., unpublished observation). Glucagon (Fig. 7) is cleaved in low yield (5-15 %) but selectively (Patchornik et aE., 1960). Solutions of glucagon in which the tryptophan residue had been oxidized by NBS still showed hormonal activity A slower oxidation and cleavage of the model peptide ethyl indolepropionylglycinate is shown by a number of reagents that are in part less
.
252
B. WITKOP
4
TRYPSIN (2.5 HOURS) CLEAVES 5 PEPTIDE BONDS TRYPSIN (50 HOURS) CLEAVES 7 PEPTIDE BONDS
t ?9,:‘,“
(2-5 HOURS) CLEAVES 5 PEPTIDE BONDS
1
(24 HOURS) CLEAVES 10 PEPTIDE BONDS NBS (LESS THAN I MIN) CLEAVES I PEPTIDE BOND
FIG.7. Multiplicity of enzymat,ic cleavages in glucagon (Bromer el al., 1957), and select,ivity of nonenzymatic cleavage with NBS (Patchornik el al., 1960). TABLE VII Cleavage of Ethyl Indole-3-propionylglycinalewith Various Oxidizing Agents0 Oxidizing agent Sodium metaperiodate in water Sodium metaperiodate in aqueous HBr Sodium iodate Sodium bromate Sodium bromate-HBr (in amounts t o form 1 mole of B r d C Sodium bromate-HBr (in amounts to form 1.8 moles of Brz) Lead tetraacetate Osmium tetroxide Periodic acid, 2 moles Iodic acid, 2 moles
Yield of ethyl glycinateb 70% 62%
67% 12% 0 76% 11% 14% 44% 55%
Gross and Witkop, 1960, unpublished data. Assayed by ninhydrin procedure. c The combination NaBrOa-HBr in a ratio t h at produces 1 mole of bromine per mole of tryptophan peptide may prove t o be useful for studies where only oxidative modification but not cleavage of tryptophan bound in a protein is required. a
b
aggressive than NBS. Table VII summarizes some preliminary observations (Gross and Witkop, 1960, unpublished data).
C. Selective Cleavage of Tyrosine Peptides (Schmir et al., 1959); Corey and Haefele, 1959; Schmir and Cohen, 1961) Bromination of a simple tyrosine analog, phloretic acid (p-hydroxyphenylpropionir acid) (CIV), with two equivalents of reagent in aqueous
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
9
Br
H O - ~-c ~ 2 c H , C O O H Brz
HO-
253
’
\
-CH,CH,COOH
Br (CIV)
(CV)
acetic acid leads to the formation of the expected 3,5-dibromophloretic acid (CV) in high yield. However, quite different results are obtained in t,he reaction of phloretic acid (CIV) with bromine or with N-bromosuccinimide (NBS in acetate buffer at pH 4.6). The course of the reaction is readily followed by measuring the rapid and extensive changes in the ult,raviolet spectrum. An oxidative reaction leads to the appearance of a very intense peak at 260 mp, the optical density attaining its maximum in 3 min with three equivalents of bromine or of NBS. When the reaction is conducted on a preparative scale in acetonitrile-acetate buffer, a neutral, crystalline product separates shortly after the addition of reagent is hegun. The same substance is obtained by the action of one equivalent of NBS on dibromophloretic acid (CVIT). The neutral product is a dihromospirodienone lactone (CVI).
3~:~
H o ~ C H 2 C H 2 c ~ H
o w Br
(CIV)
Ho+cH2CHzcmH
Br
(CVI)
(CVrr)
The course of the reaction may be considered to follow either path a or path b. By analogy to the proposed mechanism of formation of the spirolactone of dioxindolepropionic acid (XCI 3 XCIII), the concerted displacement reaction (path a) is preferred to the displacement of a C-bromo atom in a tribromodienone (path b).
path a Br
-
Br
Br
N-Acyl derivat,ives of t,yrosine and of 3,s-dihromot yrosine undergo
254
B. WITKOP
analogous transformations. N-Benzoyl-L-tyrosine (CVIII, R = CsHs) and (CX, R = CHI , N-acetyl- and N-carbobenzyloxy-3,5-dibromo-L-tyrosine Cbz) were converted in high yield into their respective N-acylaminodienone lactones (CIX) by the action of NBS. JNBS
-Ho*
-
Ho-Q--CH,CHCOO I H NHCOR
NBS
CH,CHCOOH I
-
NHCOR NHCOR
Br
(CWI)
Br
(ex)
(CIX)
When the dienone lactone (CVI) was heated at reflux with 2 N hydrobromic acid for 1.5 hr, dibromophloretic acid (CVII) was isolated in 40 % yield from the reaction mixture. The reduction process is considered to proceed according to the following scheme.
-9 OOH
0
+ JH B r H ' o Br
Br
Br
(CVI)
CH,CH,COOH
42
+ Br,
Br (CW)
This unusual reduction by halide ion of the vinylog of an a-acyloxyketone, presumably via an a-haloketone intermediate, finds analogy in the reduction of simple a-haloketones by halogen acid (Newman, 1951;Meyer, 1911; Backes et al., 1921). This interesting reductive conversion of the dienone to the dibromo derivative of the starting material was encountered in the, at the time inexplicable, cleavage of oxytocin and vasopressin (Mueller et al., 1953; Ressler et al., 1953; Popenoe and du Vigneaud, 1953;Ressler and du Vigneaud, 1954). To avoid such reductive degradation, the spirodienone lactone was refluxed with 4 N sulfuric acid for 3 hr. The crude product no longer showed any significant absorption at 260 mp and a substance, isolated in 40% yield, was determined by analysis and infrared spectrum to be a phenolic acid of composition CsHs04Brz.By fusion, the acid was readily converted into a phenolic lactone. For comparison, the isomeric dibromolactones (CXI and CXII), resulting either from oxygen migration (CXI) or from carbon migration (CXII), were synthesized. The lactone of the rearrangement product was shown t o be the resorcinol derivative (CXI) rather than that of hydroquinone (CXII) .
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
255
Br
\
(CX)
(Clm)
The ability of peptide bonds to participate in the lactonization reaction was demonstrated with phloretylglycine (CXIII) and with 3,5-dibromophloretylglycine. The addition of NBS to a solution of the peptide in acetonitrile buffer results in the formation of the dienone lactone and the liberation of glycine. The correspondence between dienone formation and Br
0
B; fCrn1)
(CXrV)
release of glycine was demonstrated by measuring ultraviolet absorption at 260 mp and by quantitative ninhydrin assay following the addition of varying amounts of NBS (Fig. 8). The cleavage reaction shows a maximal yield, based on ninhydrin assay for glycine, of at least 80%.It was found in control experiments that glycine reacts rapidly with NBS. The drop in ninhydrin color yield when NBS is present in excess of theory (Fig. 8) may be attributed to this secondary reaction. When the unusual bromine cleavage of oxytocin and vasopressin, reported by du Vigneaud and his associates, was repeated with a simplified tripeptide model, N-carbobenzyloxy-S-benzyl-L-cysteinyl-L-tyrosyl-L-isoleucine (CXV) (Ressler and du Vigneaud, 1957), bromine or NBS liberated a ninhydrin-reactive material in 40 % yield. Paper chromatography proved isoleucine to be the sole ninhydrin-reactive substance present. The ultraviolet spectrum of the reaction mixture showed the characteristic 260 mp absorption for a dienone (CXVI). Bz-SC~S-HN
Bz-SCys-Tyr-Ileu I NCbz
3
or 3 NBS
I
'cbz
w o+ isolewine Br
0 (CXV)
(CXVI)
235
B. WITKOP
More recent studies by Schmir and Coheri (1961) have led to a better understanding of the details and to improved yields in the tyrosine cleavage Phloretylglycine was oxidized with three equivalents of NBS in various buffer mixtures and the extent of cleavage determined both by ninhydrin assay for glycine and by the intensity of the dienone peak a t 260 mp in the ultraviolet. From the results (Table VIII) it is evident that cleavage is favored by increasing the acidity of the reaction mixture. I
I 1.0
I
I
2.o
I 3.0
MOLES NBSIMOLE PEPTIDE
FIG. 8. Reaction of phloretylglycine with N-bromosuccinimide: 0 , ultraviolet tlbvorption at 260 mp; 0 ,ninhydrin color based on glycine. From Schmir et al. (1959).
It is further apparent that increasing alkalinity affects release of glycine and formation of dienone to different extents and possibly in different ways. Although the spirodienone lactone is unstable in alkaline media, destruction of preformed dienone lactone by this pathway can be no more than 5 % under the reaction conditions employed. Compounds (CXVII and CXVIII) which show dienone absorption but fail to release glycine may also be formed a t higher pH values (Goodwin and Witkop, 1957; cf. Scott et al., 1957; Heine, el al., 1955; Stirling, 1960). I n addition, the extensive degradation of amino acids by positive hulogen reagents (Chappelle and Luck, 1957; Konigsberg et al., 1960; Schonberg
METHODS FOR CLEAVAGE A N D MODIFICATION OF PROTEINS
237
0
O
=
Br
I1 - CH,CH,CNHCH,COOH 9
Br
(CXWI)
(CXVII)
et al., 19Eil ; Goldschmidt et al., 1927; Langheld, 1904) must be considered
not only as a means of destroying glycine but also of consuming a significant portion of the limited amount of oxidant added. By the use of media of relatively high acidity, the oxidation of simple amino acids by NBS can TABLE VIII pH Dependence of the Cleavage of Phloretylglycinea
a
PH
% ’ Glycine
1.9 2.3 3.5 4.7 5.2 5.9 6.8 8.6
73 59 53 56 42 35 36 16
83 80 77 75 73 71 59 No peak
Schmir and Cohen, 1961.
be successfully inhibited, as shown in Fig. 9. Further, it is evident from the data in Table IX that consistently high cleavage yields may be obtained in dilute aqueous acids, including acetic, as reaction media. Whereas ortho bromination and dienone formation are competitive reactions at pH 4.6, acidic media tend to retard the latter reaction without affecting ortho hromination. These conclusions are evident from the families of curves shown in Figs. 1OA and 10B. In addition, the excellent correspondence between ninhydrin yield and dienone intensity shown in Fig. 11 indicates that, at least in acidic media, spectral intensity may be used as a valid measure of the degree of cleavage. Spectroscopic assay of reactions run in aqueous halogen acids (0.1-2 N) may be temporarily obscured by strongly absorbing polyhalide ions. For example, addition of NBS or of bromine to 1N hydrochloric acid produces an intense maximum at 235 mp, probably due to the BrClzion or to HBrClz . The absorption is rapidly discharged by a reducing agent such as phenol or formic acid or by passing nitrogen through the solut.ion. In the presence of
258
B. WITKOP
phloretylglycine the polyhalide peak decreases until the 260 mp peak for dienone emerges (Fig. 12). Spectral data for several polyhalide species are recorded in Table X.
70 v)
m
6o
Z
k 50 z 0
t
40
8
30
5u1
3e
20 10
50
100
MINUTES
Fro. 9. Effect of pH on rate of consumption of N-bromosuccinimide (0.04 2cf) by leucine (0.01 M ) . From Schmir and Cohen (1961).
TABLE IX NBS Cleavage of Phloretylglycine in Acidic Mediaa Solvent 0.016 N HC1 1.8 N HCI 0.01 N HlSOi 0.08 N HzSO, 20% Acetic acid 50% Acetic acid 80% Acetic acid a
% Glycine
% Dienone
73 80 76 84 90 89' 84
83 82 79 86 -c
-c -C
Schmir and Cohen, 1961. of several runs varying in yield from 82-96%. Spectroscopic assay obscured by high concentrations of acetic acid.
t, Average c
Experiments with a series of N-acylated tyrosine peptides indicated that cleavage yield was relatively independent of the nature of the N-acyl group preceding or of the amino acid following tyrosine (Table XI).
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
259
In order to determine the degree and rate of consumption of NBS by other functional groups in a peptide chain, a number of amino acids and their derivatives were treated with NBS, the disappearance of positive halogen being followed by thiosulfate titration. As summarized in Table
c
A
0.9
3.0 moles NES
B
0.8b 3.3 MOLES NBS
0.7 .7 MOLES NBS
0.6 2.4 MOLES NBS
0.5 >
t z u1
g 0.4 2.5 moles NBS
1.6 MOLES NBS
4I
0
I-
% 0.3
2.2 mole: NBS 1.2 MOLES NBS
0.2 PHLORETYLQLYCINE
0.I LORETYLGLYCINE
0 mF
260 200 300 320 340
h mP
FIG.10. A. Ultraviolet spectra resulting from the cleavage of phloretylglycine (lo-' M ) with successive increments of N-bromosuccinimide in 0.8 N HzSOi. From Schmir and Cohen (1961). B. Ultraviolet spectra resulting from the cleavage of phloretylglycine (9.2 x 10-6 M ) with successive increments of N-bromosuccinimide at pH 4.6. From Schmir and Cohen (1961).
XII, there is no strong competition by simple amino acids or by hydroxyamino acids in acidic media. A more rapid reaction is observed with the basic amino acids, phenylalanine and aspartic acid, and a very rapid consumption of NBS by the sulfur amino acids, tryptophan and histidine. Early experiments with the tripeptide, N-carbobenzyloxy-S-benzylcysteinyltyrosylisoleucine gave cleavage yields which were disappointingly low
260
B. WITKOP
until it became evident that the thio ether linkage was competing with tyrosine for the available NBS. From the data of Table XI11 it is apparent that both methionine and cystine offer significant competition for NBS and reduce cleavage yield. Results with the tripeptide also suggest that tyrosylpeptide cleavage is slower than sulfoxide formation but faster than that of sulf one.
I 2 MOLES NBSIMOLE PEPTIDE
3
4
FIG. 11. Reaction of phloretylglycine with N-bromosuccinimide: 0 , ultraviolet absorption a t 260 mp; 0 , ninhydrin color based on glycine. From Schmir and Cohen (1961).
Since it had already been demonstrated that tryptophyl peptide bonds can also be cleaved by NBS (Patchornik et al., 1958a, b), the competition between these two amino acids was studied via model compounds. When mixtures of equimolar quantities of phloretylglycine and indolepropionyl~~-phenylalanine (Schmir and Cohen, 1961) in ,50%acetic acid were oxidized with increasing proportions of NBS, increasing amounts of phenylalanine were liberated, as shown by paper chromatography. Glycine began to appear only after three equivalents of NBS had been added, demonstrating (at least for simple systems) a high degree of selectivity in the fission of these two peptide bonds. A preferential cleavage at tryptophyl peptide
METHODS FOR CLEAVAGE A N D MODJFICATTON OF PROTEINS
201
bonds has also been observed in more complex peptides. Thus, glucagon has been split selectively at the tryptophyl bond (Patchornik et al., 1958b, 1960) and similar results have been obtained with tobacco mosaic virus and with serum albumins (Ramachandran and Witkop, 1959). I II
m
E P
0.9
PHLORETYLGLYCINE IN I 8 N HCI S MIN AFTER ADDING NBS ( 3 a q 1 AFTER 20MIN AFTER 30 MIN AFTERSO MIh
08
or 06
>
c-
2
05
W 0 J
4
a.
04
0
0.3
02
01
00
220
260
300
FIG.12. Ultraviolet spectra resulting from the reaction of phloretylglycine with NBS in 1.8N HCl. From Schmir and Cohen (1901).
When phloretylglycine was oxidized with NBS in the presence of imidazolepropionic acid, formation of dienone was not seriously inhibited and apparently proceeded faster than attack on the imidazolc ring (Table XIV).
B. WITKOP
262
TABLEX Spectral Data for Polyhalidesa ~~
~~
Compoundb
Solvent
Species of polyhalide
Maximum
NBS or Br2 NBS or Br2 NBS or Bra NBS or Brz NBS or Brz NBS or Brz NBS or Brz NCSc
0.1 N HCl 1.0 N HC1 6.0 N HCl N NaCl 2.0 N HzSOi 2.0 N HBr N NaBr 1.0 N HC1
HBrClzd HBrClz -
End absorption 233 mp 22,000 233 mp 21,000 End absorption End absorption 265 mp 25,000 End absorption End absorption
HBra HCL
Schmir and Cohen, 1961. Concentrations = 3 x 10-6 M. c NCS = N-chlorosuccinimide. I n preference t o HBrzCl (cf. Scott, 1953). (1
b
TABLE XI NBS Cleavage of Substituted Tyros y l Peptidesa Substrate Phloretylglycinec N-Beneoyltyrosylglycine N-Acetyltyrosylglycine N-Acetylt yrosylglycylglycine 2-Tyrosylhistidined Phloretylglycine 2-Tyrosylphenylalanine .OEt 2-Tyrosylalanine .OEt 2-Tyrosylserine .OMe 2-S-Bz-Cysteinyltyrosylisoleucinef
Cleavage yield Solvent
0.01N 0.01 N HzSOi 0.01 N HzS04 0.01 N HzS04 0.01 N HzSOc 50% Acetic acid 50% Acetic acid 50% Acetic acid 50yoAcetic acid 50% Acetic acid
Ninhydrinb Dienone 76 73 73 77 -I
79
-
77 80 70
82 68 80 77 79
a Schmir and Cohen, 1961. * T h e appropriate amino acids or their derivatives were used as colorimetric standards. c Three equivalents of NBS were used in all cases except where noted. d Five equivalents of NBS. * Ninhydrin assay obscured by formation of ammonia. f Four equivalents of NBS. Isoleucine identified by paper chromatography.
It is evident that considerable ninhydrin-positive material, probably ammonia, is released in the reaction of the imidazole ring with NBS. Somewhat greater interference is shown by the imidazole ring in the oxidative cleavage of carbobenzyloxytyrosylhistidine. With the addition of three equiva-
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
263
lents of NBS, the dienone intensity is only 57 % of theory, five equivalents being necessary to reach a value of 70 %. In a preparative experiment, the dienone lactone of carbobenzyloxy-L-tyrosine was isolated in 36 % yield after the addition of a total of six equivalents of NBS. In titration experiments no measurable difference in the rate of uptake of NBS by imidazole could be detected in the pH range 1.0 to 8.0 and from spectral data it was evident TABLE XI1 Consumption of N B S b y Amino Acids and Derivatives" A Substrate Phenylalanine Aspartic acid Arginine Lysine Glutamic acid Alanine Valine Serine (threonine) Asparagine Glycine Hydroxyproline Proline Aceturic acid 2-Aspartic acid Gly cylleucine Ditosyltyrosine HistidineO
$7, Disappearance of NBS in: 15 Min
60 Min
16 13 10 11 5 4 3 3 2 1 1 0 0 1 1 2 100
36 32 30 28 17 14 12 10 7 4 2 1 0 2 2 10
-
a Schmir and Cohen, 1961. M, solvent = 0.8 N H & 0 4 containing 1% 8Substrate = lo-' M, NBS = acetonitrile (solvent for NBS addition). The following substrates consumed 1 equivalent of NBS within 2 min: histidine, imidazole, tyrosine, tryptophan, methionine, cystine. 0
that neither imidazole nor its oxidation products contributed significantly to 260 mp absorption. In another test of tyrosine-histidine competition, samples of synthetic valyl-hypertensin were oxidized with three equivalents of NBS at pH values H .Asp (NH,) -Arg-Val-Tyr-Val-His-Pro-Phe .OH
from 1.1 to 5.0. Spectral intensities at 260 mp reached values of 70-95 % of theory within 8 min. From cleavage experiments in 50 % acetic acid, yields
264
B . WITKOP
TABLE XI11 Effect of Sulfur Groups on Cleavage Yie1cP-b NBS Ninhydrin yield (%) substrate
Substrate
Addition
Phloretylgly cine Phloretylglycine Phloretylglycine Phloretylglycine Phloret ylglycine Phloretylglycinc Phloretylglycine Phloret ylglycine Tripeptidec Tripeptide Tripeptide Tripeptide Tripeptide
None None None Z-Methionirre (1 equiv.) 2-Methionine (1 equiv.) Z-Methionine (1 equiv.) Di-2-cystine (I equiv.) Di-2-cystine (1 equiv.) None None None None None
Schmir and Cohen, 1961. In 50% acetic acid, substrate c 2-S-Bs-Cys-Tyr-Ileu.
1 2 3 1 2 3 2 3 1 2 3
8 36 96 9 11
37 34 52 1 5 8 79 81
4
5
a
M.
TABLE XIV Effect of the Zmidazole Ring on the Cleavaye of Phloretylglycinea Substrate PGc PG IPC IP IP PG PG PG PG PG
+ IP + IP
+ IP + IP + IP
Equivalents NBS
yoNinhydrinb
% ’ Dienone
1.5 3 .O 0.75 1.5 3.0 I .5 2.25 3.0 3.75 4.5
9 59 93 142 136 12 44 90 167 197
10 73
11 39 69 75 73
Schmir and Cohen, 1961. Glycine standard. 0 PG = phloretylglycine, IP = imidazolepropionic acid, both 10-4 M in 0.01 N HCl . b
METHODS FOR CLEAVAGE AND MODIFICATION
OF PROTEINS
265
of DNP-valine up to 15% (uncorrected for losses in hydrolysis or elution from paper) were obtained. The selective cleavage of the six tyrosine peptide bonds in ribonuclease is described under Section V, E.
D.Selective Cleavage of Methionine Peptides Sulfonium salts derived from methionine may decompose in a variety of ways depending on the conditions employed and the nature of the S-alkyl groups. Electron-withdrawing groups on the sulfur atom facilitate breakdown of the sulfonium salt by intramoleuclar or bimolecular nucleophilic displacement. Toennies and Kolb (1945b) suggested that the decomposition of N-formylmethionine methylsulfonium acetate to homoserine lactone on evaporation of an aqueous solution to dryness in vacuo occurred through a n internal displacement of dimethyl sulfide by the carboxyl group. A similar decomposition was not observed with the sulfonium halides. Decomposition of the sulfonium salt derived from bis-(2-chloroethyl) sulfide and methionine by heating an aqueous solution for several hours a t 100°C yielded largely methionine and a small amount of homoserine (Stein and Moore, 1946). The heat-labile principle of cabbage juice, which is more effective than molar equivalents of rnethionine in preventing the toxicity of sulfanilamide for Escherichia coli, was found to be a methionine methylsulfonium salt (McRorie et al., 1954). Autoclaving a solution of methionine methylsulfonium iodide in water yielded homoserine. Boiling the methionine methylsulfonium salt, isolated from asparagus, with alkali yielded homoserine with liberation of methyl sulfide (Challenger and Hayward, 1954; Lavine et al., 1954). In the decomposition of methionine methylsulfonium ion in acid solutions methionine is regenerated, whereas in hot neutral and alkaline solutions homoserine and methyl sulfide are formed. The decomposition of S-adenosylmethionine (CXIX) a t p H 4 or in neutral solution gives nearly quantitative yields of methylthioadenosine
k
N $ >
b
._ -.. -.
a;pH 4-7
a t 100°C o r A. aei’ogenes
‘\, ‘\, \
Q
CH-CHOH-CHOH
CH+CH+
s--cH~--cH,- CHNH,
L---O---q
,/’ chemically I
I
only chemical split: ting by alkali known
ips
stable, enzymatically mobile (XCIX)
/coo@
enzymatic decarboxylation preliminary to transfer of propylamino group
266
B. WITKOP
and homoserine (Cantoni, 1953; Parks and Schlenk, 1958a, b) . Homoserine lactone has also been observed among the products of similar hydrolysis and is not produced from homoserine under these conditions (Parks and Schlenk, 1958a). Enzymatic decompositionof S-adenosylmethionineby cellfree extracts of Aerobacter aerogenes produced methylthioadenosine and homoserine lactone (Shapiro and Mather, 1958). It was later proven by incubations in tritiated water that the S-adenosylmethionine-cleavingenzyme of baker's yeast produces homoserine lactone directly by an intramolecular displacement and not via 2-amino-3-butenoicacid (Mudd, 1959). The presence of homoserine, homoserine lactone, and S-carboxymethylhomocysteine among the products of acid hydrolysis of ribonuclease, 50 % inactivated by iodoacetate at pH 2.8, and the poor recovery of methionine provides another example for this type of breakdown of methionine sulfonium salts (Gundlach et al., 1959b). The same products in varying proportions were observed in the decomposition of methionine carboxymethylH,CS
H
0
H.+ HZC, ,C=O
bw,
Methionine
H,C-$ H,C-:O
H Ie
Carboxymethylsulfonium salt of methionine
H,C-0 I
S-Carboxymethylhomocysteine
I
Homoserine lactone
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
267
sulfonium iodide at 100°C at various pH's (Gundlach et al., 1959a). The original interpretation was that the homoserine lactone arose from homoserine and not directly by an internal displacement reaction. In view of these many precedents it is surprising that the intramolecular displacement of the sulfur function of methionine derivatives has not been utilized for selective cleavage of methionine peptides. The method has only recently been applied t o a number of methionine peptides which according to the following scheme (CXX-CXXIII) were first converted to sulfonium salts and then subjected to intramolecular (imino) lactonization and hydrolysis by short heating in water (Lawson et al., 1961). Table XV shows the strong influence of the nature of the added alkyl group on the yield in THS TH2 I ICH2CONH2 35 - 4 0 ° C
0 CH2 COOH
@CI-A
CH,
'1
S
CHz-CONH2
7% .TI1 R-CH-NH I
NH-C-R'
COOH
0) CHZ I C 4 H 0
I
II
NH-C-R'
(CW
yHS
S-CCH&ONH,
COOH
t
R--CH-NH,.?iI
I
COOH
+ NH-C-R' (CXWI)
the subsequent cleavage of the sulfonium salt. Best yields (53%) were oht ained with the strongly electron-wit hdrawing carb amylmet hyl (-C HzCO-NH,) group.
268
B. WITKOP
The procedure was applied to additional dipeptides, and the course of the alkylation at 3540°C followed by argentometric titration (Toennies and Kolb, 1945a). With over 90 % formation of carbamylmethylsulfonium salts only 8 % of peptide cleavage occurred at 40°C; however, on brief heating a t 95OC the yields of liberated amino acid increased to 54-85 % (Table XVI). Paper chromatography and paper electrophoresis of such reaction mixtures showed that in each case the liberated amino acid was the only ninhydrinpositive substance present. TABLEXV Injluence of A l k y l Group on the Cleavage of Sulfonium Salts Derived f r o m Ethyl N-Acetylmethionylglycinate~
Alkylating agent
Iodoacetic acidd Methyl iodidedg * Ethyl bromoacetated 1 8 Iodoacetamidef
2,4-Dinitrofluorobenzenef Diethyl bromomalonatef
Molar concentration of peptide in reaction mixtureb 0.005 0.01 0.01 0.01 0.01 0.01
Equivalents of alkylating
agents 3 4 4 3
4 3
Percentage of peptide cleavagec
6.0 3.6 43 53 2 5
Lawson et al., 1961. Reactions were allowed t o proceed a t room temperature for 24 hr. c After removal of excess of alkylating agent by ether extraction the reaction mixture was heated for 1 hr a t 100°C. The liberated amino acid was determined by ninhydrin assay (Moore and Stein, 1948). Reaction medium was 0.1 M , pH 3 citrate buffer. 8 Alkylation was conducted in a sealed tube. f Reaction medium was 1:l mixture of EtOH and 0.1 M , p H 3 citrate buffer. b
Since in peptides and proteins alkyl halides a t pH 2.8 rcact only with the sulfur of methionine (Gundlach et al., 1959b), this procedure permits specific chemical cleavage of methionyl peptide bonds. To determine whether the sulfonium acetates and/or nitrates lead to better yields than the iodides in peptide cleavages such salts of the methionine peptides were prepared and cleaved concurrently with the corresponding iodides. The iodides, acetates, and nitrates with and without heating before analysis gave comparable cleavage yields (Table XVII) (Lawsoti rt al., 1961). A marked improvement in the cleavage of methionhe peptides was achieved by the use of cyanogen bromide (Gross and Witkop, 1961). Prc-
TABLEXVI Alkylation and Cleavage of Methionyl Peptides" Time of alkylation (hr)
Peptide
Degree of alkylation by Percentage Of titration (%) peptide cleavage
Carbobenzoxy-L-methionyl-Lglutamic acidbv 0
68d 135f 135f 135f
92 91 91 91
8.1. 800 85h 81i
Benzoyl-oL-methionylglycineb *
68d
99
8.66
Benzoyl-m-methionylglycine ethyl ester* c
135' 135, 135,
93 93 93
54g 65h 62'
Car bobenz oxy-L-met hionyl -Ltyrosineb
6sd 65d
92 84
84h
7.9'
Lawson et a t . , 1961. Concentration in ethanol-water (1:l) was 2 X 10-2 M . c Three equivalents of alkylating agent were used. Temperature of alkylation was 40°C. 0 Mixture was fractionated on columns of Amberlite IR-120 and the cleaved amino acid was determined with automatic recording equipment (Spackman et aE., 1958). f Temperature of alkylation was 35-40°C. Reaction mixture was analyzed directly by ninhydrin method (Moore and Stein, 1948). Reaction mixture was heated for 1 br at 95°C before analysis (Moore and Stein, 1948). i Reaction mixture was extracted with ether, heated for 1 hr a t 95"C, and analyzed (Moore and Stein, 1948). b
Comparison o j Yields
TABLEXVII Cleavage of Iodides, Acetates, and Nitrates of Methionine Sulfonium Peptidesa i 7 ~the
~~
Per cent of peptide cleavageb Peptide
Carbobenzyloxy-L-methionyl-L-glutamic acidd Benzoyl-m-methionylglycine ethyl esterd Carbobenzyloxy -L-methionyl-L-tyrosined
Iodide
Acetate
Nitrate
Ana- Heatedc lyzed before directly analysis
Ana- Heatedc Iyzed before directly analysis
Ana- Heatedc lyzed before directly analysis
79
87
a2
81
81
86
40
63
46
66
47
68
74
92
77
85
77
85
Lawson et a l . , 1961.
* Determined by ninhydrin
assay for the cleaved amino acid. One hour at 95OC. d Alkylation mixture was ethanol-water (1:l by volume) solution which was 0.02 M in peptide and 0.06 M in iodoacetamide. Alkylation temperature was 40°C. Alkylations were 9@-100% complete by titration. c
269
270
B. WITKOP
viously von Braun et al., (1923; 1926; 1931) had used this reagent for the
cleavage (CXXV, CXXVI) of thio ethers. The reaction which may be conveniently formulated as a four-center process (CXXIV) requires elevated temperatures for completion. Intermediate cyanosulfonium bromides have not been isolated. By contrast cyanogen bromide reacts with ethyl N-benzoyl-m-methionylglycinate in aqueous alcohol at room temperature because of the intramolecular assistance in the displacement of methylthiocyanate (CXXVII) to yield, via the unstable cyanosulfonium salt, (a) 70 % N-benH,C--Q:- -CN
COOEt
CH.SCN
COOEt
zoyl-DL-homoserine lactone (m.p. 143"C, reported 142°C) (Fischer and Blumenthal, 1910), (b) methylthiocyanate, assayed by gas chromatography and infrared absorption, and (c) 75-90 % of ethyl glycinate. The application of this highly selective chemical cleavage method to bovine pancreatic ribonuclease will be described in Section V. The involvement of methionine sulfonium salts in these chemical cleavage reactions may stimulate experiments to show the significance of special methionine residues in the active centers and the catalytic sites of enzymes. Such an important role for a particular methionine has been postulated for the enzymes phosphoglucomutase and chymotrypsin on the basis of photooxidation studies (Ray el al., 1960).
E. Cleavage of Histidine Peptides
I I
The -C=Cdouble bond of the imidazole ring in histidine is in the allylglycine position and, theoretically, should invite participation of the C-peptide group in suitable displacement reactions (CXXIX).
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
271
(CxXrX)
This is indeed the case. Table XVIII summarizes preliminary results (Shaltiel and Patchornik, 1961) on the reaction of some histidine peptide TABLEXVIII Cleavage of Histidine Peptides by N-Bromosuccinimidea Moles of NBS for required maximal cleavage
Histidine peptide
N-Carbobenzyloxy -L-histidinylglycine N -Carbobenzyloxy-L-histidinyl-mphenylalanine Ethyl-N, N'-dicarbobenzyloxy-L-histidinylglycinate Methyl-N ,N'-dicarbobenzyloxy-L-histidinyl-L-leucinate Methyl-N,N'-dicarbobenzyloxy-L-histidinyl-DL-phenylalaninate CeHa
Liberated amino acid
3 3
Glycine Phenylalanine
2
Ethyl glycinate
2
Methyl-L-leucinate
1
Methyl-m-phenylalaninate
CHr-CH-COCsHs
I COHN-C / HN I
' A
AH
0-
\
NH-CH2-C
0 OH
(Product of Bamberger cleavage of histidine)
COCeH5
a
Shaltiel and Patchornik, 1961.
derivatives with NBS in aqueous acetic acid. The yields were determined by ninhydrin assay and semiquantitative paper chromatography; they ranged between 15-35%. Maximal yields were obtained with 3 moles of NBS for N-carbobenzyloxyhistidine, 2 moles of NBS for N ,N'-dicarbobenzyloxyhistidine peptides (Patchornik et al., 1957) and 1 mole of NBS for the peptide attached to the fragment resulting from hydrolytic Bamberger cleavage (Bamberger, 1893; Kossel and Edlbacher, 1915; Witkop and Beiler, 1956) of histidine ester under Schotten-Baumann conditions. These results support the involvement of the y ,&double bond in the
272
13. WITKOP
cleavage. This double bond, part of a dibenzoylaminoethylene in the product of the Bamberger cleavage, is &ill reactive and requires only 1 mole of NBS. Whether participation of the peptide group involves the usual bromonium intermediate (CXXIX) is still ail object of further studies. I n the latter reaction a y-lactone derivative has been obtained whose structure is under investigation (Shaltiel and Patchornik, 1961). The action of N-bromosuccinimide on imidazole itself, in aqueous media, leads to rupture of the ring as well as to the formation of tribromoimidazole. The cleavage products have been identified as glyoxal, ammonia, and probably formate (Cohen and Schmir, 1961). Yields of glyoxal up to 40 % have been obtained, using 1 M equivalent of halogenating agent. With substituted imidazoles, such as imidasole-4-propionic acid or N-acyl histidines, an analogous oxidative cleavage occurs and the corresponding a-ketoaldehydes have been isolated as quinoxaline derivatives in yields up to 60%,, with one equivalent of oxidant. When the imidazole ring is blocked by formation of N-toluenesulfonyl derivatives, the attack by NBS is prevented or greatly retarded. Only in the case of imidazoles with a propionic acid side chain (CXXX) does the blocked ring show reactivity. Although NBS is rapidly consumed, no ammonia is liberated. Participation by the side-chain carboxyl to form a bicyclic derivative is believed to occur. When the side chain is present as the propionamide, N-benzylpropionamide, or propionyl glycine, NBS is again consumed without liberation of ammonia, benzylamine, or glycine (Schmir and Cohen, 1961). Although the nature of the reaction product is still under investigation, infrared spectra of the crude products suggest, the possibility of a bicyclic compound (CXXXI -+ CXXXII).
Another possible aspect of histidine peptide cleavage is introduced by t,he great lability of 4(5H)-imidazolones (CXXXIII) (Freter et al., 1957; Brown and Kies, 1959). In their lability and eagerness to open up to formamidino acids (CXXXIV) they almost resemble anhydrides (Kny and Witkop, 1959). It is not unlikely that such 4(5H)-imidazolones can be produced nonenzymatically. Such selective oxidation of a hist,idine peptide to a 4(5H)-imidazolone derivative introduces the possibility of trans1act)a-
METHODS FOR CLEAVAUE AND MODIFICATION OF PROTEINS
(CXXXV)
273
(CXXXVI)
mization (CXXXV) with the N-peptide bond to an N-acylpyrrolidone (CXXXVI) and further hydrolysis to a new COOH-terminal fragment. At the present exploratory level the cleavage of histidine peptides has to await further refinement in order to be applicable to proteins and to qualify as a selective cleavage.
F. Cleavage of Simple y-Aminobutyryl and y-Glutamyl Peptides Glutamine (cf. Wieland and Pfleiderer, 1957) has been notorious for giving anomalous amounts of free nitrogen in the van Slyke determination. Instead of only 50%, all of its nitrogen is liberated, unless proper care is exercised to keep the concentration of nitrite low. The carbonium intermediate (CXXXVII) invites participation of the amide, forming the iminolactone (or possibly the hydrated aminolactol) which hydrolyzes to 4-carboxy-y-butyrolactone (CXXXVIII) and either ammonia or nitrogen depending on the concentration of nitrite ion (Austin and Howard, 1959). A similar iminolactone intermediate is formed from y-aminobutyrylglycine
(CXXXVII)
(CXXXV-III)
(CXXXIX) on treatment with nitrous acid, to judge from the breakdown
‘0’
:~~_CCE~H,COOH
2,
I ..___ _-_...-‘IN H,C /cH, HONiO+ H L ..._.-_. ?! H, (CxXxrX)
+ [H,NCH,COOH]
(CXL)
274
B. WITKOP
products, free glycine and y-butyrolactone (CXL) (Francis and Witkop, unpublished data). This kind of cleavage is probably of practical interest only in small peptides which contain y-aminobutyryl or y-glutamyl residues. The yields are moderate (20-40 %) and depend markedly on the conditions. For example, the classical elucidation of the structure of glutathione (CXLI) made use of a deamination reaction with nitrous acid (Quastel et al., 1923). The reaction mixture containing deaminated glutathione did not contain any
ether-soluble product. This means that a-hydroxyglutaric acid or its lactone is not formed by spontaneous hydrolysis of the intermediate iminolactone (CXLII) but rather by vigorous acid hydrolysis of the spirothiazolidine (CXLIII) formed by direct (arrow b) or subsequent addition of the thiol group (CXLII CXLIII) to the neighboring immonium ether (arrow a ) . Deamination of primary aliphatic amino groups in (suitably substituted) peptides may also be possible via diazonium fluoborates and triazenes (White and Scherrer, 1961):
I----
Cleavage of peptides (?)
The reaction should lend itself in principle to fission of those peptides that are cleaved by nitrosating agents. This cleavage method will probably be of value in the case of the interesting novel peptide antibiotic Cephalosporin C (CXLIIIa) (Abraham and Newton, 1'361)
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
Ha$
275
\ /s CH--(CH~)~-CO-NH-CH--CH b H z I I / I
eooc
OC-N
I
TCHz-OCOCH8 COOH
(CXLIIIa)
which has been converted to 7-aminocephalosporanic acid in small yield by hydrolysis (dotted line) with dilute acid (Loder et al., 1961).
G . Attempted Cleavage of 0-Tosylhydroxyproline Peptides I n the attempted tosylation of N-aroyl derivatives of 2-amino-2-methyl1-propanol not the 0-tosylates (CXLIV) but corresponding 4,4-dimethyl2-aryl-2-oxazolines (CXLV) were obtained.
I Ar
I Ar
(CXLIV)
(CXLV)
Likewise one might expect 0-tosylation of N-tosylhomoserylglycine (CXLVI) to yield the iminolactone which would break down into N-tosylhomoserine lactone (CXLVII) and glycine.
NHTs (CXLVI)
(CXLM)
N-Acylated 0-tosylhydroxyproline derivatives (CXLVIII) are known t o undergo intramolecular displacement of 0-tosylate anion by the carboxylate anion under mild conditions leading to N-acylated lactones of allohydroxyproline (CXLIX) (Patchett and Witkop, 1957). The application of this intramolecular displacement principle to amides and peptides of N-acylated 0-tosyl- or 0-mesylhydroxyproline derivatives (CL) has not met with success so far (Francis and Witkop, unpublished
data). The reasons for this failure may be twofold: (a) the bicyclic [2,2,1]y(imino)lactone (CLI) is strained and forms with less ease than a monocyclic y-lactone ; (b) the intramolecular displacement of tosylates of secondary alcohols by neighboring amide groups may require either S N l or S,2 conditions (cf. Bly and Dryden, 1959), and meets with steric and inductive hindrance in a five-membered pyrrolidine system. Conditions that are
e.
c=o
P
7
"
Q R
k
(CXLIX)
(CXLVIII)
0 II CNHCH,COOH
I
0
Qp~NHCHPCOOH
///
Ts (CIA)
known to favor elimination and the formation of olefins, such as refluxing of N-(-O-ditosy1)hydroxyprolylglycine in dimethyl sulfoxide (Nace, 1958) did not, lead t o cleavage nor was amide interaction observed in dimethyl formamide (Chang and Blickenstaff, 1958). Systematic studies on the possibility of elimination reactions with O-tosyl- and O-mesylhydroxyproline derivatives or attempted dehydration of N-acylated hydroxyprolines have all met with failure. This approach was abandoned when in peptides made from synthetic 3,4-dehydro-n~-proline (Robertson and Witkop, 1960) the double bond proved to be uncooperative in participation reactions involving the action of N-bromosuccinimide or strong acid. Strong acid is known t o cause iminolactonization and cleavage of amides (Craig, 1952) and peptides (Lawson and Witkop, 1961a) of e.g., 2,2-diphenyl-4pentenoic acid. The oxidation of bound hydroxyproline (CLII) to a 4-ketoproline peptide (CLIII), reduction by sodium borohydride to the allohydroxyproline peptide (CLIV), and the action of acid might lead to cleavage (CLV-CLVII)
<
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
277
which would be preferential rather than selective and thus not be readily applicable to the cleavage of hydroxyproline peptide bonds in collagen (cf. Patchett and Witkop, 1957). 0
m -l-
~
OH
JR
0
c
N I co
I
(CLII)
N
A0
I
(CLIII)
I
(CLVII)
I
(CLW
I
I
co
I
co
I (CLVI)
co I
(CLV)
V. SELECTED APPLICATIONS A . Tryptophan Titration The drop in extinction at A 280 mfi which accompanies the oxidation of tryptophan to an oxindole derivative offers a method for measuring the accessibility of tryptophan and the content of accessible tryptophan in a given protein. The use of an empirical factor of 1.31 to multiply the actual decrease in absorption and of the molar extinction coefficient of 5500 for tryptophan a t 280 mp makes possible the calculation of tryptophan present. Figure 13 shows these changes in extinction for the NBS titration of tryptophan in bovine serum albumin (Ramachandran and Witkop, 1959), which is dissolved in 10.0 M urea solution in order to make the tryptophan units accessible. Another convenient way of picturing the changes in extinction is shown in Fig. 14 (Peters, 1959). Here one recognizes at a glance that rihonuclease contains 110 tryptophan. Based on a value of 2.8 X for the amplitude in drop of molar absorption of free tryptophan it was concIuded that human serum albumin (HSA) contains one, bovine serum albumin (BSA) two, and ovalbumin probably four rather than three tryptophan units.
278
B. WITKOP 0.7
t
0.5
v)
z W
a
J
a 2
k 0
0.3
0.1
240
280
240
320
WAVELENGTH,
280
mp
FIG.13. (A) Bovine serum albumin (1.65 mg) in 1.55 ml of 10.0 M urea solution at pH 4.15 (curve 1) ; after addition of 0.25 p M of NBS in 25 A of water (curve 2) ; after addition of 50 X (0.5 p M ) of NBS (curve 3). No further decrease in OD280 with addition of NBS was observed. (B) Human serum albumin (Hg dimer, 2.5 mg) in 3.03 ml 10 M urea solution a t pH 4.15 (curve 1) ; after addition of 0.2 p M of NBS in a 1 pM/ml solution in water (curve 2); 0.3 p M of NBS (curve 3); 0.45 fiM of NBS (curve 4) ; 0.65 p M of NBS (curve 5) ; 0.75 p M of NBS (curve 6). The original spectrum was practically unchanged after the addition of only 0.1 p M of NBS. From Ramachandran and Witkop (1959). 0
4
AE:
0 -4
-0
-12
0
10
20
30
40
NBS, MOLEIMOLE FIQ. 14. Change in millimolar absorbance of tryptophan and various proteins on reaction with progressively increasing amounts of NBS. Figures in parentheses indicate the reported number of tryptophan residues per mole of protein. Conditionv: 4-12 X M albumin, 8 M urea, pH 4.0. From Peters (1959).
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
279
The changes in the ultraviolet spectrum of creatine phosphokinase (Fig. 15) on treatment with NBS point to the presence of thirteen to fourteen tryptophan residues per molecule compared with eleven to twelve tryptophan residues suggested by chemical analysis (Friedberg, 1956). Although the oxidation of the tryptophan residues in the molecule seems t o be complete, the amount of cleavage of peptide bonds next to tryptophan is very small.
140
120 100
80
60 40 20
0 240
250
260
270
280
290
300
310
320
FIQ.15. Absorption spectra of creatine phosphokinase in 8 M urea-0.1 M acetate buffer, pH 4.0 after treatment with diffcrent amounts of NBS. Numerals indicate moles of NBS per mole of protein. Samples were read azainst a urea-bufferblank containing identical concentrations of NBS. From Quiocho et al. (1961).
The tryptophan ccntents of various proteins as determined by the NBS titration method is shown in Table XIXa. The determinations of tryptophan may be done at pH 4 in aqueous formate-acetate buffers, in 8 M lithium acetate-acetic acid, 70 % acetic acid, or 8 M urea-acetic acid. Only with the hemoglobin (Satyanarayana Rao, 1961) has there been a sizeable difference in the analytical values obtained in aqueous formate-acetate buffer versus 8 M urea-acetic acid, suggesting the presence of a few “buried” tryptophyl residues which have to be unmasked by urea before oxidation can take place. Four moles of
280
Il. WITKOP
TABLE XIXa Detervnination of Percentage of Tryptophan in Various Proteins b y “Titration” with N B S or NBAa Tryptophan content (%) Protein NBS “titration”
Glucagon” TMV proteinb I-peptideb Lysozymeb Gramicidin Chymotrypsina Chymotrypsinogenc Trypsinogene Trypsine Human serum albuminb Bovine serum albuminb Hemoglobini Human Cow Sheep Rat Creatine phosphokinasek Thiostrepton’
2.12 (2.3, 2.8, 3.08) 3.62 8. 3 42.8 5.7 5.7 3.4 3.3 0.21 0.51 1.97 1.88 2.57 2.36 0.33-0.35 16.53‘
Literature value
Total amount of NBS or NBA consumed for maximum decrease of indole absorbance at 280 mp; moles/ mole tryptophan
5.71 2.6-3.2 2.2-3.1
2.6 -
3.6-4.41 7.1, 9.1. 45d 5.7’ 5.69 3.7h 4.5, 1.oi 0.19 0.58
1.95-3 .O 2.3-3 .O 1.7
2.00“
6.4 5.5 4.7 6.0 17.2 1.5-1.8
-
0.25-0.3 15.83’”
-
1.8 3.2 10.0
Cf. Patchornik et al., 1960. Cf. Ramachandran and Witkop, 1959; Peters, 1959. Fromageot and de Garilhe, 1950; Lewis et al., 1950. d Synge, 1949. Viswanatha et al., 1960; Viswanatha and Lawson, 1961. f Weil et al., 1953. 0 Northrop et al., 1948. h Keil and gorm, 1954. Block and Bolling, 1951; Block and Weiss, 1956. i Satyanarayana Rao, 1961, R Quiocho et at., 1961. Ramachandran, 1961; cf. Bodanszky et al., 196Ob. m This value was obtained by Rarnachandran (1961) by the Goodwin and Morton (1946) method. So far t#hereis no proof that the “indole component” of thiostreptin is identical with tryptophan or with the recently discovered derivative of 8-hydroxyquinoline (Drey at al., 1961). Rossi-Fanelli et al., 1955. 0
b
tryptophan per mole is found in aqueous buffer in sheep and rat hemoglobin, whereas in 8 M urea-acetic acid values of 8 and 9 residues per mole, respectively, are obtained. Differences in reactivity of particular residues to NRS should, when possible, be compared with results from other tech-
281
METHODB FOB CLEAVAGE AND MODIFICATION OF PROTEINS
TABLEXIXb Cleavage of C-Tryptophyl Bonds i n Peptides and Proteins Material
Glucagonb
TMV proteinc
Bond cleaved (yield in %)
Me diumo
Try-Leu (8) Try-Leu (6) Try-Leu (14) Try-Ala (35); TryLYS (29) Try-Ala (28); TryLys (23) ; Try-Thr
A B A 13
C-terminal heptadecapeptide from TMV proteind Pentadecapeptide from TMV protein0 Bovine serum albuminc
Human serum albuminc Ovalbuminc LysosymcC
NBA
NBS 5
3.5
-
-
Try-Ala (10) Try-Ala (19) Try-Ala (18) Try-Thr (10-20)
B B B A A
3.3 -
Try-Lys
A
-
(4)
I-peptidec
Moles reagent per mole tryptophan
C Try-Ser (2245) ; TryGly (38-68) Traces B C Try-Ser (30); TryG b (50) C Try-Ala (1640) C None C Try-Asp, Try-Val or Met,. Try-Ser, TryAla Try-Val (33), TryA Phe (8), Try-Leu (IS), Try-Arg (6) Try-Gly (15), TryC Val (5), Try-Ser (2), Try-Phe (1) Try-Val (45-60), Try- B “Leu” (38-65), Try-Arg (26-351, Try-Gly (1633), Try-Ser (24-32)
10, 8-20
5 10-20 5 8-15
5
66
a Symbols : A-Aqueous acetate-acetate-formate, pH 4; B-70Y0 acetic acid; C8 M urea-acetic acid, pH 4. b Patchornik el al., 1960. c Ramachandran and Witkop, 1959; Peters, 1959. Gish, 1959a. Bernier and Jollbs, 1961. 6
282
B. WITKOP
niques like the spectrophotometric solvent perturbation technique (Laskowski and Herskovits, 1961) for location of “buried” and “exposed” tryptophyl groups (cf. Section V, C). There may indeed be degrees of reactivity in residues depending on the secondary and tertiary structure of the protein. In general values obtained by NBS oxidation agree well with the reported values in the literature. In the “titration” (Table XIXa) and cleavage (Table XIXb) experiments both NBS and N-bromoacetamide (NBA) have been used. The oxidation by NBS of tryptophan in proteins and peptides, as judged from spectral changes, is almost always instantaneous during gradual addition, while with NBA, within the ratio of 2 moles to one of tryptophan, as long as 20 min are needed before the absorbance at 280 mp reaches a steady minimum. TABLE XIXc Destruction of Side-Chain Amino Groups of Lysozyme by NBSa as a Function of N B S Concentration and Oxidation Medium cDNP-Lysine found, moles/mole Protein
Moles NBS/mole tryptophan
In 0.2 M Acetateformate, PH 4
Lysozyme
0 1.6
4. 6 3.9 3.6
2.5 5.0 a
2.7
In 8 M LiOAc, pH 4
Theoretical
4.6
6
2.7 2.5 1.4
Ramachandran and Witkop, 1958.
Peters (1959) found, however, that decrease in absorbance at 280 mp in urea with proteins (bovine and human serum albumins, ovalbumin, lysozyme) was not instantaneous, but required 20-60 min depending on the amount of NES added, while other spectral changes occurred within one minute (cf. Section V, B). In cleavage experiments a large excess of NBS is to be avoided, because of the risk of unwanted side reactions. In the cleavage of tryptophyl-glycine derivatives in acetate buffer excess reagent capable of destruction of liberated glycine (Patchornik et al., 1960) was counteracted by formic acid which is rapidly degraded (Barakat et al., 1955) in the process to COZ, HBr, and succinimide. The extent of side reactions will depend on the nature, number, and proximity of free or potential functional groups. The loss of c-NH2 groups of lysine in lysozyme under various conditions is illustrated in Table XIXc. With bovine mercaptalbumin and hemoglobin A which t)ake up approximately 5 and 20-30 moles reagent, rcspectively, before decrease in
283
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
absorbance at 280 mp is detected the oxidation of S H groups (one and six S H groups, respectively, are present in the two proteins) may be the most rapid reaction. The oxidation of S H to HSO3- requires 3 moles of reagent. Varying degrees of oxidation of S H and SS-groups have been observed depending on amount of reagent used (Table XIXd) .Drastic conditions of reaction may yield quantitative oxidation of 44- bonds. TABLEXIXd Cysteic Acid Content of Hydrolyzates of Proteins Oxidized with NBA i n Aqueous Acetate-Formate Buffer at pH 4 Protein ~
Content
Ribnuclease
::;zi
Insulin
___-
Number of CyHSO, eatimated Number of S atoms present aa half-cystine Amount of NBA used, moles/ mole protein a
2.0 8
4.9 10 (9-8) (S-S)
33
42
1.2 6
(S-S) 27
t;gi
1 Gkzn 1 I Bovine
Hemo- TMV globin protein /'-peptide
-_____ 4.2 10 (S-S)
17.8 30
(1SHand
s-€3)
32
124
-~
7.2 6
0.17
(-SH:
(-SH)
1
0.11 1
(SHor 8-S)
81
Ramachandran and Witkop, 1969.
TABLE XIXe Products of Oxidation of Di-DNP-Cystine by NBAa
Material
Di-DNP-cystine Di-DNP-cystine Di-DNP-cystine
Medium
Formate-acetate buffer (0.4 M) pH 4 SO% AeOH Water
Products formed as moles per cent Mole ratio NBA added DNP-cysteic DinitroAcid aniline 10
72.5
-
10 10
64.6 73.1
26.9 5.2
Ramachandran, 1961.
The relative ease of oxidation of a model disulfide, di-DNP-cystine, is illustrated in Table XIXe, where the amount of oxidant used was just in 50 % excess (Ramachandran, 1961). The yield of 73 % DNP-cysteic acid is not far from values for yields of cysteic acid (80-90 %) from performic acid oxidation of cystine (Schram et al., 1954). Proteins with reactive 4 H , S-S-, or free -NH2 groups therefore pose problems when side reactions in the oxidative cleavage of peptide bonds is to be avoided (cf. Section V, B).
284
B . WITKOP
B. Selective Cleavage of Tryptophgl Peptide B o n d s in S e r u m A l b u m i n s As the titration curves for the various albumins show, a considerable amount of NBS is consumed before a steady decrease of 6 at 280 mp sets in. Apparently other reactive groups, especially thiol groups, react faster with NBS than tryptophan in this case. Figure 16 shows that after reaction with about 10 moles of NBS per mole of HSA (molecular weight -65,000) only one major new NHAerrninal residue, namely alanine, is liberated which was determined by the standard
o'8k 1.0 I
AMINO DNP- 0.6
ACIDS, 0.4 M./M.
0.2
OO
-
10
20
NBS, M./M. FIG.16. N-Terminal amino acid residues detect,ed in human serum albumin after treatment with varying amounts of NBS. Yields of DNP-amino acids were corrected for lomex on hydrolysis. From Peters (1959).
DNPAMINO 0.6 ACIDS. 0.4 0.2 $0
20
30
40
SO
60
NBS. M /M
FIG.17. N-Terminal amino acid residues detected in bovine serum albumin after treatment with varying amounts of NBS. Yields of DNP-amino acids were corrected for losses on hydrolysis. From Peters (1959).
DNP procedure. The yield of this cleavage approximated 50 % assuming that the 0.8 mole of DNP-aspartic acid represents one peptide chain. Likewise BSA which contains two tryptophan units on treatment with 5-15 moles of NBS per mole of protein (molecular weight 435,000) liberated only two new NHz-terminal residues, namely serine and glycine in yields of 60 % and 50 %, respectively, in addition to the original NHZ-terminal aspartic acid (Fig. 17). The selectivity of these tryptophan cleavages in the albumins amounts to splitting of one or two peptide bonds among about 560. With the present technique the usefulness of the method for the albumins is restricted t o rapid spot checking of sequence changes next to tryptophan
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
285
as a function of species variations or genetic mutations. No fragmentation and separation into characterizable subunits as a result of NBS treatment has hem observed on attempted fractionation with trichloroacetic acid, ammoiiium sulfate, ultracentrifugation, slid electrophoresis. Apparently the split portions of the chain are still securely held together by secondary or tertiary linkages. Of particular interest in this connection is the controlled fragmentation of the y-globulin fraction from serum which at neutral p H is surprisingly stable to enzymatic attack. Preliminary results show a highly destructive effect of NBS even at 0°C and p H 5 for only 20 min in the absence of urea or detergent after only one-fifth of the tryptophan residues reacted (Porter, 1960). A more recent ultracentrifugal analysis (Phelps et al., 1961) of a sample of human y-globulin which had been reacted with 0.01 M N-bromosuccinimide indicated a t least two components the sedimentation constants of which were 5.36 and 2.34 S after correction for the density and viscosity of the urea solution. A second sample reacted with 0.05 M N-bromosuccinimidr had a single component but gave no evidence of more slowly sedimenting material. The ultraviolet absorption spectrum of the brominated samples was completely changed in accordance with reported observations. No maximum was discernible at 280 mp. It is difficult to conclude whether or not the new slowly sedimenting components represent fragments similar to those formed in the oxidation reaction since the effect. of urea on the sedimentation behavior of the brominated protein cannot be estimated. The new selective cleavage of methionine peptide bonds with cyanogen bromide offers more promise of success. There are about twenty methionine residues in y-globulin and three to four methionines in the 50,000 molecularweight fraction which has retained the power to bind antigen (Porter, 1957).
6. “Buried” and “&xposed” Tryptophan in Tobacco Mosaic Virus ( T M V ) Protein The primary sequence of the TMV protein as it has been established recently by the Tubingen (Anderer et al., 1960a) (Fig. 18) and Berkeley (Fraenkel-Conrat, 1960) groups contains three tryptophan units. However the NBS titration method applied to TMV protein (A-protein) led to a decrease of optical density a t 280 mp commensurate with only 1.9 moles of tryptophan per mole of protein of molecular weight 18,270. After the reaction of 6 moles of NBA per TMV protein subunit 0.2-0.3 mole each of alanine and lysine were released as new NHz-terminal residues. The so-called I-peptide with 3 moles of NBA per mole of tryptophan in 66% acetic acid or with 3 moles of NBS per mole of tryptophan in aqueous 0.2
286
B. WITKOP I
ACETY L-SERlTY R-SER-ILEU-THR-PRO-THR-SER-FLU-PHE~VAL-PHESLEU~SE C
P
C
P
41
6
?LA1 LEU TGLY-+SP-$,LUFPHE~~LU-THRT~LUTFLU~ALA?ARG-THR~VAL-~LU.~VAL, C
P
P
C P
NH2 NH2P
C NH2 P
CNYCNH2C P
P
C P
NH2P
c
46
71
68
PHE-PRO-ASP-SER-ASPiPHEiLYS PC
P
92
1
VAL-TYRTARG TYRT~SP-ALA?VAL?ASP~PRO 6
C 6 C N PH 2
P
P
P
P
P
90
P
P
P
P
100
ILEU-~LUTVALTGLU j+3P-GLU-ALA-+SP-PRO-THR-THR-ALA~GLU~THR-LE~SP NH2 P
PNH2 kH2
N Y
P
P
112
LEUTALA-VALyTHR-AL A-A SP-ASP-VAL-A P
ILEU~ASP-LEUTILE U-VAL-GLUTLEU- I ‘P
CNHz
$
b
P
P
134
A P
-VAL?LEU-GLY-SER-SER-SER-GLU?PHE-SER-SER C
C P
C P
FIG.18. Primary sequence of protein from tobacco mosaic virus protein (Anderer et al., 1960a). The multiplicity of enzymatic cleavages is indicated by C (chymotrypsin), P (pepsin), and T (trypsin). Cleavage by NBS is observed next to Try (17), Try (52), and to a lesser extent next to Try (152). From Ramachandran and Witkop (1959).
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
287
M sodium acetate-acetic acid buffer at pH 4.0 containing 0.2% sodium dodecyl sulfate led to the cleavage of a peptide bond and the detection of 0.2 mole of alanine as new NHz-terminal. The attempted isolation of the two fragments has been reported to be unsuccessful (Anderer et al., 1960a, b). Although reaction conditions have not been reported, it seems that destruction of the tertiary structure after oxidative cleavage, e.g. by brief heatdenaturation, may be needed before a separation of the two fragments will be noticeable. The intact I-peptide itself has a marked tertiary structure and ability to form large aggregates as revealed by electron microscopy (Fraenkel-Conrat and Ramachandran, 1959). The pentadecapeptide (KO.,peptide) with 3 moles of NBS per mole of tryptophan was found to yield 0.4 mole of lysine as NH2-terminal (Gish, 1959a, 1960; Wittmann and Braunitzer, 1959). These data indicate the presence of Try-Ala and TryLys bonds in TMV protein. The absence of any other major N-terminal group after cleavage with excess NBS originally supported the assumption that there are only two tryptophan residues in this protein (cf. FraenkelConrat and Ramachandran, 1959). From the absence of N-terminal arginine and aspartic acid one may conclude that peptide bonds attached to tyrosine carboxyls are not split under the conditions used, since two of the four tyrosine sequences in TMV protein are known to be Val-Tyr-Arg- and Gly-Tyr-Asp-NH2 . Not until the COOH-terminal tryptic hexadecapeptide became accessible in a pure form (Gish, 1959b, 196l), has it been possible to ascertain the presence and full reactivity of a third tryptophan unit by the NBS method. The new NHz-terminal released after cleavage is threonine. Chart I (Ramachandran and Witkop, 1959) summarizes these results: The two sequences Try-Ala (17-18) and Try-Lys (52-53) are “exposed” and fully reactive NBS; the sequence Try-Thr (151-152) is “buried” (cf. Herskovits and Laskowski, 1960) in the A-protein, but becomes accessible to NBS in the tryptic hexadecapeptide. In addition to the methods of spectral perturbation of chromophoric groups by polyhydroxy compounds, spectrophotometric titration studies, and ultraviolet difference spectra the reactivity of tryptophan toward selective agents such as NBS may offer a new tool for assessing influences of secondary or tertiary structure in this area. In a given protein inaccessibility of tryptophan units to NBS may not necessarily mean stability to enzymatic cleavage. In TMV-protein, for instance, all three tryptophan peptide bonds are cleaved by the action of chymotrypsin. This means that the approach of a chemical cleaving agent and of an enzyme is affected by the same enviromental factors to a different degree. Exclusive iodination of the A chain of insulin is a related example of an unreactive tyrosine residue in the B chain (Springell, 1961).
CHART I.-NBS
f3 00 00
CLEAVAGE OF TMV PROTEIN AND PEPTIDES
TMV-VI R US
l+iiq J
JI TMV-PROTE I N (A-PROTEIN)
RIEONUCLEIC ACID (RNA)
158 AMINO ACIDS
I 17 52 152 158 N-Ac-SER-TYR.-TRY--TRY...TRY--TRY*.*THR 2 NBS:
TFR-ASPJ I
J.
\1
PRECIP. p H 4 . 9
15 AMINO ACIDS(47-61)
41 AMINO ACIDS (1-411
I
47
41
N -Ac- SER-..TRY-ALA--ARG
1-1
NBS:
YIELD 20%
\1
k 0.7-PEPTIDE
I-PEPTIDE
61
GLU--*TRY-LYS--ARG I NH2
NBS:
(TRY-LYSI
YIELD 20% 3 NBS/MOLE TRY
C -TERM I NAL PEPT IDE 17 AMINO ACIDS (142-158) I42
158
SER.-TRY-THR-.ALA-THR
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
280
Ramachandran recently extended this work and determined the tryptophan content and tryptophyl sequences of proteins from six different plant viruses by the NBS titration as well as other methods (Table XX) (Ramachandran, 1960). The results of the NBS cleavage of the tryptophan peptide bonds in the various virus species are summarized in Table XXI. An observation of some interest is the antigenicity of the I-peptide (41 amino acids) from TMV protein. These antigenic properties are not lost, TABLEXX Tryptophan Content of Six Virus Proteins" Tryptophan Protein sample from strain of TMV
c---+---.,.-+,....
Masked GA (green aucuba) YA (yellow aucuba) J14D1 (yellow aucuba) HR (Holmes rib grass) Cucumber virus 4
3.43 3.39 3.27 2.99 2.73 1.20
(%I
Literature valuesb on NBS oxidation Microbio- Colorlogicalc imetryd
.____
(3.03)~ (2.99) (2.95) (2.64) (2.41) (1.05)
3.54 3.41 3.49 3.71 1.78 1.21
(3.12)e (3.01) (3.07) (3.26) (1.56) (1.06)
2.2 2.1 2.1 2.2 1.4 0.5
4.3 4.2 4.2 4.4 3.5 1.4
Ramachandran, 1960. Values in the last two columns are for the viruses and will need to he multiplied by a factor 100/94 t o obtain values for the proteins. c Knight, 1947. d Knight and Stanley, 1941. The colorimetric values reported in the literature are by the glyoxylic acid reaction. a Values in parentheses represent moles of tryptophan per mole of protein, assuming a common peptide-chain molecular weight of 18,000.
after treatment with an amount of NBS small enough to lead only t o oxidation of the one tryptophan present in the peptide, presumably without appreciable cleavage (Bozicevich et al., 1959). Such observations raise the possibility of use of NBS in the modification of proteins for immunochemical purposes such as the preparation of toxoids. At the present time relatively unspecific reagents, such as formaldehyde or p-propiolactone, are used for this purpose.
D . Cleavage of an Antibiotic Cyclopeptide, Gramicidin A The intramolecular participation of the C-peptide group in the iniinolactonisation reaction of tryptophan peptides requires a certain freedom
290
B. WITKOP
from steric strain and interference. The naturally occurring antibiotic cyclopeptides tyrocidin A and tyrocidin B were of special interest because of possible steric restriction of the iminolactonizatioii reactions of the TABLE XXI Yield of N-Terminal Residues on NBS Oxidation“ * b * Protein sample from strain of TMV Masked
GA (green aucuba)
YA (yellow aucuba)
J14D1 (yellow aucuba)
Conditions of reaction (28”C, 1 hr) 70% AcOH, 3 moles NBS/mole tryptophan
Lys 0.22 Ala 0.06 Thr 0.15 Lys 0.09 Ala 0.04 Thr 0.05
70% AcOH, 6 moles NBS/mole tryptophan
6 M urea-AcOH, pH 4, 3 moles NBS/mole tryptophan
Lys 0.04 Ala trace T h r trace
Lys 0.06
-
-
Lys 0.03
-
Nil
-
T h r 0.07 (Ser 0.03, Val 0.06, “Leu” 0.07, artifact 0.01)
Lys 0.17 Ala 0.13 T h r 0.19 (Ser 0.01)
Lys 0.12 Ala 0.11 T h r 0.14 (Traces of Asp and artif act)
Lys 0.05 Ala 0.01 Thr 0.06
Lys 0.08 Ala 0.10 Thr 0.03
-
-
H R (Holmes rib grass)
Ala 0.08 Thr 0.06 (Artifact 0.12)
Cucumber virus 4
T h r 0.06 (Ser. 0.01) ~
~~~
Ala 0.04 T h r 0.04 (Trace artifact) Ala 0.07 Thr 0.12 (Artifact 0.12)
-
Lys 0.05 Ala 0.15
Nil
Nil ~~-
~~~~~
Ramachandran, 1960. Values are expressed as number of moles per mole of protein of weight 18,000. The entry “artifact” refers t o a spot, in the Levy two-dimensional chromatogram of DNP-amino acids, occurring in the region of phenylalanine and whose identity has not been established. a
tyrosyl or tryptophyl peptide bonds. The release of new NH2-terminalresidues after the reaction of tyrocidin A or B with NBS under comparable conditions was smaller than in model peptides. Of more practical interest is the NBS cleavage of gramicidin A which
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
Om-Leu-D-Phe
/
Val 1 TYr
h r o
‘Gh -Asp-D-Phe Tyrocidin A
he
291
P-Leu--D-ph LI o
I Val
TYr hu4sp-D-Phk
Try
Tyrocidin B
contains over 40 % of tryptophan in an unknown sequence. Its cleavage has always presented great difficulties because the molecule is stable to the action of enzymes, even very active bacterial proteinases (Ishii and Witkop, 1961) such as the one from Streptomyces griseus (Nomoto et al., 1960). The high tryptophan content piecludes the use of acid for hydrolysis. Basic hydrolysis is unsuitable because of the accompanying racemization. Gramicidin A, however, is attacked by N-bromoacetamide (NBA) and N-bromosuccinimide (NBS) (Gross and Witkop, unpublished observation). In 50 % aqueous ethyl alcohol at room temperature 5 % of the peptide bonds (20% of the tryptophyl peptide bonds) are cleaved with NBS. Methyl alcohol must be avoided because it opens the spirodioxindole lactone from oxidized tryptophan to the ester even at room temperature. The cleavage mixture separates on electrophoresis (pH = 2.5, sodium-formate buffer) into four ninhydrin-positive components of which the fastest migrating one was identified as ethanolamine. Dinitrophenylation showed leucine and danine to be additional NHrterminals of the released fragments. It has been possible to increase the cleavage yield notably by reacting gramicidin in buffer solutions (pH = 4.5) of high lithium acetate concentrations (Patchornik et al., 1960) with either N-bromoacetamide or N-bromosuccinimide (Gross and Witkop, unpublished observation). Lithium ion is removed from the solution as the sulfate. After evaporation of the ethyl alcohol the water solution contains mainly ethanolamine while the other fragments are water-insoluble and precipitate. This precipitate in a ternary system of chloroform/acetic acid/water (100:75 :25) was separated into = 255 mp) and ninhydrin-positive comthree ultraviolet-absorbing (A, ponents (Chart 11),the NHz-terminal of fragment I being leucine. It has been found recently that gramicidin A carefully purified by countercurrent distribution (cf. Craig, 1949) separates into two to three components on thin-layer chromatography (Ishii and Witkop, 1961).
E. Selective Cleavage of the Six Tyrosine Peptide Bonds i n Ribonuclease Crystalline ribonuclease (Fig. 19) was chosen as a model for the selective cleavage of t,yrosine peptide bonds in more complex polypeptide systems (Cohen and Wilson, 1961). Although on addition of NBS the ultraviolet spectral intensities at 260 mp reached 80% of theory, cleavage of the six
292
B . WITKOP
CHARTI1 Partial" Purijcation of Commercial Gramicidinb by Countercurrent Distribution and Cleavage by N-Bromoacetamide 8
CCD: 999 transfers, 40/40 ml upper and lower phases Syst : 15 C6Hs , 15 CHCI, , 23 MeOH, 7 HzO Type of gramicidin Yields* Distribution coefficients
I
I'
Bb
A
2%
25%
= o.30
K
=
I
0.56
I
CC 3%
Dc 0.5Y"
K = 1.5G
K = 16.65
1) NBA
7.25 M LiAcOH-EtOH ( l : l ) , p H = 4.5, room temp., 20 min 2) HISO,, EtOH
' 1
LizSOa
Supernatant I
Evaporation of EtOH
I
Precipitate
Supernatant : ethanolamine
CCI): 200 transfers, 10/10 nil upper and lower phases Syst: 1.00 CHCI, , 0.75 CHICOOH, 0.25 HzO
Yields+
I
I
I1
I11
23%
3%
45%
1
FDNU
DNP-LEU ~-
~
Tests for the homogeneity of these fractions by special chromatographic techniques are in progress. Ishii and Witkop, 1961. Gregory and Craig (1948). Grow and Witkop (unpublished). The yields refer to the peak fractions only.
tyrosyl peptide tmiids, determined by quantitative dinitrophenylation, fell short of expectations (Table XXII) . The negligible splitting of a tyrosylhalf-cystine bond is of particular interest, since simple derivatives of tyrosylhalf-cystine gave at least 80 % cleavage. Improved yields were observed
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
t CHEMICAL TAIL
293
PEPTIDE----IB”~~
FIG. 19. Topography of the NBS cleavage of the six tyrosyl peptide links of native and S-carboxymethylribonucleafie (Cohen and Wilson, 1962) and topography of the cyanogen bromide cleavages of the four methionyl peptide bonds in native ribonuclease [simplified diagrammatic “approximation” of Spackman et al. (1960)l. Studies at t h e National Heart. Institute and The Rorkefeller Institute for Medical Research on the order of residues 11-18 are now essentially complete and will he published shortly (personal communication from the Editors of Advances in Protein Chemistry). TABLE XXII Cleavage Yields of Ribonucleasea Amino acid
Native RNaseb
(%)
(%I
Lysined Glutamic acid Serine Valine Cysteine Proline
22 57 84 27 26 54
31 34 65 60 33f
CMRNasec
209
Determined by dinitrophenylation; values corrected for destruction during acid hydrolysis (Cohen and Wilson, 1962). * I n 50% acetic acid, using 46 equivalents of NBS. Carboxymethylribonuclease, in 50% acetic acid, using 50 equivalents of NBS. In addition to the original N-terminal lysine of RNase. 8 As cysteic acid. f As S-carboxymethylc ysteinesulfone. 9 Determined as the phenylthiohydantoin.
in the oxidative splitting of S-carboxymethylribonuclease, in which the sulfur function is oxidized t,o a carboxymethylsulfone by NRS rather than to a sulfonic acid. All four tyrosine bonds of oxidized insulin have been cleaved b y the ac-
294
B. WITKOP
tion of bromine water, and the liberated new NH2-terminals confirmed the presence of Tyr-Glu and Tyr-CyS03H of the glycyl chain, and of Tyr-Leu and Tyr-Thr of the phcnylalanine chain (Thompson, 1960b).
F . Selective Cleavage of the Four Methionyl Peptide Bonds in Ribonuclease and the Separation of the Fragments The usefulness and selectivity of the cyanogen bromide method for the cleavage of methionyl peptide bonds (cf. Lawson et al., 1961) was demonstrated with ribonuclease (Gross and Witkop, 1961), which in a chain of 124 amino acids contains four methionines (Hirs et al., 1960; Hirs, 1960). Bovine pancreatic ribonuclease, specially purified by countercurrent distribution according to Gregory and Craig (1948) was allowed to react in 0.1-0.3 N HC1 solution with up to 30 equivalents of cyanogen bromide a t 20°C for 24 hr. After removal of solvent, excess reagent, and methylthiocyanate by lyophilization the residue in aliquots of 1.5-2.0 mg was subjected to paper electrophoresis for 4 hr at 1100 volts, 60 mA, and p H 6.5 in a pyridinc acetate buffer system. In order of increasing electromobility the following four major fractions were identified: (I) free homoserine resulting from free homoserine lactone; (11) “core” material together with ribonuclease (possibly containing methionine sulfoxide which is resistant to NCBr) ; (111) 25 % ’ (theoretical) of isolated “chemical tail peptide,” in contrast to the enzymatically produced 20-residue S-peptide (cf. Vithayatahiland Richards, 1960), containing the original NHz-terminal lysine and a C-t,erminal homoserine (lactone) ; (IV) free homoserine lactone, by cleavage of the methionyl-methionyl-lysine (29-30-31) sequence; its yield together wit,h the free homoserine with which it equilibrates in aqueous solut,ion approximates 50 %. On a preparative scale the separation of the fractions after cleavage is best done on a column of Sephadex G-50 (Porath and Flodin, 1959) (Fig. 20), using 0.2 N acetic acid (Rasmussen, personal communication) as the eluting agent. In this procedure (Fig. 20) the core material (yield 80%) comes off first as a major and a minor peak, (probably differing only with regard to amide groups of glutamine or asparagine residues). The chemical tail peptide (yield 83%) is eluted next as a sharp peak as assayed by the ninhydrin met,hod. This tail peptide contains no tyrosine and therefore has no ultraviolet absorption at 280 mp (Fig. 19). The last fragments t o be eluted in a single fraction are homoserine and its lactone (yield 85 %). Figure 21 shows the previously published sequence of residues 11-18 of ribonuclease (Hirs, et al., 1960). Previous studies (Red field and Anfinsen, 1956; Anfinsen et al., 1956) have suggested that residue 11 is glutamic acid rather than serine. The results with cyanogen bromide cleavage discussed above are consistent with the latter assignment for position 11. Thus cleav-
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
1.50 (00.7$-
295
RIEJONUCLEASE t CNEr SEPHADEX G-50 l 9 0 X 4 C M . : 0 . 2 N CH3COOHI
1.00
-
(0.5d-
"CHEMICAL TAIL PEPTIDE"
0.50 (0.25)-
50
I00
I50
2 0
NUMBER OF 5ml. FRACTIONS
FIG. 20. Fractionation of cyanogen bromide-treated ribonuclease on a column of Sephadex G-50. From Gross and Witkop (1961).
,.---. I
ARG
' ! I
FIG. 21. The published sequence of ribonuclease in the region consisting of residues 11 through 18 (Hirs, Moore and Stein, 1960).
age following the methionine residue originally assigned position 17 in Fig. 21 should result in the formation of an NHz-terminal glutamic acid residue. However no DNP-Glu was detected after dinitrophenylation of cyanogen bromide-treated RNase. On the other hand, DNP-Asp was found, suggesting the presence of the sequence, Met-Asp, in the enzyme (Gross and Witkop, J. Bid. Chem. in press). The S-peptide of subtilisin-modified ribonuclease (Richards and Vithayathil, 1959) was cleaved with cyanogen bromide and the fragments separated by electrophoresis in three different buffer systems. The peptide
29G
R . WITKOP
fragment released by cyanogen bromide cleavage migrates at p H 3.6 and 6.5 to the anode and is the slowest one a t pH 1.9. It contains Ala, Asp, Ser, and Thr. A peptide of such composition could not be released by cleavage following the methionine residue in the sequence shown. Thesc
TABLE XXIII Survey of the NHz-Terminal Residues Involved i n the Cleavage of Ribonuclease b y Cuanoaen Bromide" Dinitrophenylation and hydrolysis Fraction Products Original mixture of reaction of cyanogen bromide with ribonuclease
NHz-term. mid. Foundb Calc.
Di-DNP-Lys
0.90
2.0
13NP-Ser
0.85
1.0
DNP-Aspc
0.3
1. 0
Numerical position and sequence of methionine(s) involved in cleavage
Met-Lys (Lys) 30 31 (1) Met-Ser 79 80 Met-Asp ?
__
Electrophoretic fraotion 11: ribonuclease (Meth-0) and modified core
lactone ~~
Di-DNP-Lys
0.75
2.0
DNP-Ser
0.65
1.0
l)NP-Aspc
0.3
1.0
Met-Lys (Lys) 30 31 (1) Met-Ser 79 80 Met-ARp ?
Electrophoretic fraction 111: chemical tail peptide (1-13)
?
Met -Met-Lys 29 30 31
DNP-Homoser
Di-DNP-Lys
0.55d
1.0
?
(Orig. NHn-terniinal Lys)
Grosv and Witkop, 1961. Corrections for losses during hydrolysis were made with authentic DNP-amino acids which were taken concomitantly through the entire procedure. c For the Rolveiit systcm used in the separation and assay of DNP-Asp, see Biserte e t al. (1959). d Hydrolyclis time was 18 hr compared with 8 hr and 90% Di-IJNP-Lys, see Arifinvep et al., (1954). a
b
results, therefore, further support the conclusion that the sequencc in 1Gg. 21 is incorrect as written. A study of the reaction of cyanogen bromide in 0.3 N HC1 with the common amino acids showed that, besides methioninr, only cystrine (cf. Swan, 1958), but neither cystine, tyrosine, nor tryptophan, reacted. Cleavage of
297
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
the a-chain of human hemoglobin containing two methionines (Fig. 22) (Hill and Konigsberg, 1960; Braunitzer et al., 1960) has been observed and led to three fractions on a Sephadex G-25 column (Konigsberg and Hill, 1961). The cyanogen bromide cleavage of tryptic fragments of the a-chain has led t o selective cleavage in yields up to 80 7% (Braunitzer, G., personal e Ser u Pro ~ i ASP a 4 s
__
marp
a
voi v
P
Val Hie Leu Thr Pro Glu Glu Lys Ser Alo Val Thr Alo Leu Try Gly Lyr Val Asp Val
26
~
Val LYI A I A ~ I ~Try GIY
32
~ y Val r G ~ Y Aia His Ala GIY
~
G I ~Tyr GIY
A I ~G I ~ ~-
-
vv Asp Glu Val GIy Gly Glu
00
40
a
P
Ala Leu Gly Arg Leu Leu Thr Glu Pro Val Val Tyr Try Are Pho Phe Glu Ser Phe Gly Asp Leu Ser Thr Pro Asp Ala Val -~ 27 50 40 so--64 70 7 7
a
P LIB
80
70
60
82
78
a
P
His Cys Asp AspAsp Thr Ser Glu Glu -Pro
83
86-102
v A l a Val v - o - L e u
Leu Leu Phe Arg Leu A l p 103
106
a
P
Gly Val Leu Leu His His Cys Ala Val Val Leu Phe Gly Lys Glu Phe Thr Pro Pro Val Glu Ala Ala Tyr Glu
a
A10 Ser Val Ser Thr Vol Leu Thr Ser Lys Tyr Arp
P
Ala Gly Vol Ala Alo Asp Leu Alo His Lys Tyr His
LYS
Val VOl
-
showing the locations of the 3 methionine residues, respectively. From Braunitzer et al. (1961a); Konigsberg et al. (1961).
communication). The reaction of hemoglobin with cyanogen chloride has been studied previously in another connection without suspicion of cleavage (Aldridge, 1950).
G. Cleavage of the His-Thr bond in the Hemopeptide of Mammalian Cytochrome c with N -Bromosuccinimide In a histidine peptide containing no tryptophan and no tyrosine NBS may be used t o advantage for the cleavage of the peptide bond next to
298
B. WITKOP
histidine as exemplified in the following (Dus and Patchornik, 1961). Horse heart cytcchronie c was digested with pepsin to yield a hemopeptide after purification as described by Tuppy and Paleus (1955) ; Margoliash et aE., (1959). The pure hernopeptide has the following sequence: Val-Glu(NH2)-Lys-Cys-Ala-Glu(NH2)-Cys-His-Thr-Val-Glu
The iron was removed from the peptide by oxidation with performic acid and the resulting peptide after purification over a column of talcum powder was treated with NBS (Saltiel and Patchornik, 1961) at p H 2, for 20 min at room'temperature. Excess NBS was destroyed by the addition of formic acid. The reaction mixture was dinitrophenylated a t pH 8.5, and the DNPpeptides were hydrolyzed with HCl. After resolution by two-dimensional paper chromatography DNP-Val, DNP-Thr, and DNP-Thr-Val-Glu were identified. The latter was formed due to incomplete hydrolysis of the peptide bond next to valine. The extent of cleavage of the His-Thr bond was found to be 20 %.
H . Sdective Modification of Enzymogen-Enzyme Pairs The schematic representation of the activation of trypsinogen to the active enzyme trypsin (cf. Dixon et al., 1958b) assumes that with the loss of the hexapeptide in the process of activation a change in secondary structure cccurs that brings groups needed for catalytic activity into close proximity in the active center of the enzyme. One would expect this change to be reflected in different degrees of reactivity, e.g., of the four tryptophans in trypsinogen versus trypsin. This was found to be the case (Viswanatha et al., 1960). I n order to achieve the same degree of oxidation of tryptophan more NBS was required for trypsin than for trypsinogen (Fig. 23). A qualitative explanation may be that trypsin after rearrangement or contraction in the process of enzyme activation has become more compact and the four tryptophan units have become less accessible than in the precursor. The oxidation of trypsin and trypsinogen was carried out in aqueous 0.1 il.1 acetate buffer solutioiis at room temperature. In this particular case and under these conditions no significant cleavage of peptide bonds next to tryptophan residues occurred. Careful analysis of hydrolyzates of NBSoxidized txypsinogen and trypsin confirmed the selectivity of the oxidative modification of the protein, as Table XXIV shows. There is no significant loss of tyrosine, histidine, serine, threonine, or cystine, although all of these amino acids will react with NBS but considerably less rapidly than tryptophan. Trypsinogen exposed to p H 4 but not subjected to the action of NBS
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
299
showed the typical activation phenomenon on treatment with trypsin st pH 8. Treatment of the zymogen with NBS however, altered the activation characteristics, the nature of the change being dependent on the amount of NBS used. Figure 24 shows that increasing quantities of NBS progressively lowered the extent of maximal activity. With 1.0, 1.5, 2.0, and 2.5 moles of NBS per mole of zymogen, maximum activities corresponded tJo60, 45, 30, and 3 % of the control. In addition to the decrease in activity, NBS treatment also resulted in a retardation of activation, dependent on the amount of NBS used. This lag in activation was also observed on very
moles NBSlmolc Tryptophan I mole8 NBSlmoIe Enzyme 4
2 8
3 12
FIG.23. Comparison of the effects of N-bromosuccinimide on the disappearance of tryptophan absorption in trypsinogen and trypsin. From Viswanatha et al. (1960).
limited oxidation (0.1 to 0.5 mole of NBS), although the same final activity as in the control was obtained. Likewise, studies on the alkali consumption during the activation of such trypsinogen samples revealed this delay. The catalytic amount of trypsin added to initiate the activation process cannot be affected by NBS, since all of the reagent is quantitatively consumed at the time of addition. Succinimide, the end product formed from NBS, was found to have no effect, either on the activation process or on the activity of trypsin. The possibility of the formation of a competitive trypsin inhibitor during the oxidation is eliminated, since oxidized trypsinogen does not interfere with the activation of intact trypsinogen. The appearance of activity in the oxidized trypsinogen samples parallels the proteolysis during activation, followed by the uptake of alkali. The lag apparently is related to the protein becoming less susceptible to the proteolysis essential to ac-
300
B. WITKOP
tivation. Aggregation of the partially oxidized zymogen is a possible explanation for the retardation of the proteolysis. The relationship between the oxidation of tryptophan and the maximal TABLE XXIV Amino Acid Composition of Trypsin and Oxidized Trypain" * Number of residues per 24,000 gm of protein Amino acids
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalaniiie Lysined Histidine Ammonia Arginine
Trypsin
Oxidized trypsin
Ohserved
Probable
Observed
Probable
22.0 9.6 32.4 14.2 8.20 25.0 14.2 11.5 11.8 1.20 12.3 14.1 9.0 3.02 13.7 3.10 29.3 2.05
22.0 10.0 32.0 14 .O 8.0 25.0 14.0 12.0 12.0 1.o 12.0 14.0 9.0 3.0 14.0 3 .O 29.0 2.0
22.2 9.7 31.7 13.9 7.9 25.1 14.2 11.4c 11.9 1.25 12.4 14.0 8.5 2.93 13.8 3.10 29.0 2.1
22.0 10.0 32.0 14.0 8.0 25.0 14.0 12.0 12.0 1.o 12.0 14.0 8-9 3 .O 14 .O 3 .O 29.0 2.0
Viswanatha et al., 1960. Salt-free trypsin (Worthington Biochemical Corporation Lot No. 68SSF) i i n oxidized, and oxidized as described in the text, were hydrolyzed by constant-boiling hydrochloric acid for 20 hr a t 105°C. Except for the oxidation step, the samples were treated identically. The oxidized sample possessed 3% of the original activity. Tho hydrolyzates were analyzed for amino acids using the Automatic Amino Acid Annlyzer (Beckman-Spinco) developed by Spackman et aE. (1958). Ninety-nine and 97% of the applied nitrogen was recovered in the case of unoxidized and oxidized trypsin hydrolyzates respectively. c A very small amount of cysteic acid (0.07 mole/24,000 gm of protein) was observed in the chromatogram of oxidized trypsin hydrolyzate. d One of t h e fourteen lysines by the use of enzymatic degradation and longer columns has recently been identified a8 erythro-6-hydroxy-L-lysineby Viswanatha and Irreverre (1960). a
6
activity is shown in Fig. 25. The two curves were obtained in experiment,s in which the protein concentrations differed. In general, it was found that the more concentrated the protein solution, the greater the effect of a given
METHODS FOR CLEAVAGE AND MODIFICATION
I
I
I
I
OF PROTEINS
I
I
I
100-
I
-
0 No NBS
I
301
80
v 4
$ 40
20
TIME (hours)
FIG.24. Influence of various amounts of N-bromosuccinimide (moles/mole of eymogen) on the time and extent of autoactivation of trypsinogen a t 25"C, borate buffer pH 8, and 0.02 M Caw. From Viswanatha et al. (1960). IOC
, t\ - 1
I
TRYPSINOGEN
8C C
.-
5 .-
6C
c
8 *-
g
P
4c
c3
5 .g
2c
5"
20 O/O
40
60 60
0
Tryptophan Destruction
FIG.25. Two representative runs (different volumes and concentrations) on the effects of progressive oxidation of tryptophan on the final enzymatic activity of trypsinogen. From Viswanatha et al. (1960).
302
B . WITKOP
amount of NBS. However, Fig. 25 shows that nearly 18% of the tryptophan can be oxidized without much effect on the activatability, which falls off rapidly with the oxidation of a second tryptophan. The influence of NBS on acetyltrypsinogen was also investigated. It resulted in a stepwise destruction of the activity obtained on peptic activation. Treatment of the acetylzymogen with 1.0, 2.0, and 3.0 moles of NBS led t o values of 25, 13, and 3% compared with the activities in control ex-
% Tryptophan Destruction
FIG.26. The decrease of enzymatic activity of trypsin a8 a function of the diaappearance of the tryptophan chromophore caused by NBS. From Viswanatha e t al. (1960).
perimeiits. In this case the oxidation of tryptophan could not be followed spectrophotometrically because of precipitation of protein. When a 0.1 % solution of trypsin in acetate buffer a t p H 4 was treated with increasing quantities of NBS, no significant effect on the enzymatic activity was noticed up to 3.0 moles of NBS per mole of protein. Further oxidation inactivated the enzyme. With 4.5 and 7.0 moles of NBS, 65 and 92% of the original activity was lost. Even after treatment with 10 moles of NBS, about 2 % ’ of residual activity was still detectable. The relationship between the oxidation of tryptophan and the loss in tryptic activity is shown in Fig. 26. This curve is similar to that for trypsinogen (Fig. 25). I n studies on the hydrolysis of p-nitrophenyl acetate (NPA) by active
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
303
and partially NBS-inactivated samples of trypsin, the activities towards NPA roughly paralleled activities towards benzoyl-L-arginine-ethyl ester (BAEE) . However, the “initial burst” (Stewart and Ouellet, 1959) of p-nitrophenol was not observed, and this was the case with all samples, even the untreated control, which was merely exposed to a 0.1 M , pH 4 acetate or phosphate buffer. With oxidized insulin as substrate, a progressive drop in activity of oxidized trypsin samples was observed. In all cases, the extent of cleavage was restricted to the two known sites for tryptic cleavage in the insulin molecule. The optimal pH for BAEE hydrolysis was not changed on partial oxidative inactivation of trypsin. The incorporation of phosphorus. Enzyme preparations from oxidized trypsinogen of varying degrees of hydrolytic activity were allowed to react with diisopropylfluorophosphate (DFP) and the amounts of incorporated phosphorus determined. Irrespective of the activity values reached after complete activation, 1 mole of phosphorus per mole (24,000 gm) of protein was incorporated. A partially activated sample, having approximately 3 % activity as compared with 25 % on maximum activation, contained only 0.17 mole of phosphorus. After complete activation this value increased to 1 mole (Table XXV). Oxidized trypsin samples, inactivated to various degrees, were also reacted with DFP. Samples of low tryptic activity still bound 1 mole of phosphorus per mole of protein. A sample which had 10 % activity incorporated 0.37 mole of phosphorus after 6 hr of reaction with DFP, but a sample with only 3 % activity incorporated 0.61 mole after 18 hr (Table XXV) . The reaction with DFP was carried out under conditions so as to minimize the phosphorylation of the second reactive site in the protein (Viswanatha, 1957). The oxidative inactivation is not reversible at pH 8, under the condition wed for phosphorylation, since suitable controls which were kept at the same pH failed to show any increase in activity towards BAEE on prolonged standing. End-group analysis. Qualitative analysis for N-terminal groups on an NBS inactivated sample of trypsin showed only one major DNP-amino acid spot, corresponding to DNP-isoleucine, from the known N-terminus of trypsin (Davie and Neurath, 1955). However, the failure to find any other spots in this experiment does not rule out the formation of minor amounts of new N-terminal groups due to tryptophyl peptide cleavage (Patchornik et al., 1958a, b; Ramachandran and Witkop, 1959); it only indicates that under the inactivation conditions extensive cleavage does not occur. N-Terminal analysis after treatment of trypsinogen with 12 moles of N-bromoacetamide in 70 % acetic acid yielded DNP-amino acid spots correspond-
304
B . WITKOP
ing to DNP-phenylalanine (about 20 %) and di-DNP-lysine (about] 3 %), in addition to the DNP-derivative of valine, the known N-terminus of trypsinogeri (Ilavie arid Neurath, 1955). Conclusions. Treatment of trypsinogeii arid trypsin with NBS results in the largely selective oxidation of the tryptophan residues in these proteins, TABLE XXV The Incorporation of Phosphorus on DFP Treatment of Samples of Trypsinoyen and Truvsin after Oxidation with NBS" No.
Enzyme
1 2 3 4 5 6 7
Trypsinogen Trypsinogen Trypsinogene Trypsin Trypsin Trypsin Trypsin
of Per cent NBS per tryptophan destruction
g:ki$, 0 1.5 2.0 0 3.G 8.0 6.0
0 17 30 0 36 77 62
Per cent activity observed" 100 50 3-5; 25,
100 50 3 Approx. 10
per mole Of phosphorus of protein
1.04 (1.06)d 0.98 (0.98) 0.17 (0.18) (1.07)' 0.90 (0.92) 1 . 0 (0.91) 0.610 0.37h (0.37)
Viswanatha et al., (1960). The molecular weight of trypsinogen and trypsin is assumed t o be 24,000. In the case of trypsinogen activation was obtained in the presence of catalytic amounts of trypsin. d The values i n parentheses represent ratios in which the protein values were obtained by spectrophotometric methods by t h e measurement of absorbance a t 280 mp and the use of empirical conversion factors, while for t h e other values the protein figures were determined by nitrogen analyses. When a large volume of a 0.2%solution of trypsinogen was treated with 1.5 and 2.0 moles of NBS, 24 and 48 hr, respectively, were required t o obtain the maximal activity (50% and 25Yo). The DFP inhibition was studied with NBS-oxidized trypsinogen st two different intervals of ttctivation. f DFP treatment of fractions with only 3-5'37" submaximal activity resulted in a n incorporation of 0.17 mole of phosphorus, which increased t o 1.07 moles a t maximum activity (25%). 9 The time of reaction with DFP was 20 hr. The time of reaction with DFP was 6 hr. a
as is revealed by the spectral changes, and supported by amino acid analyses (Table XXIV). The relative resistance of the tryptophyl residues in trypsin is probably a manifestation of over-all stmctural differences between the zymogen and the enzyme. h'early 18 and 25 % of the four tryptophan residues in the zymogen and the enzyme, respectively, can he dcstroyed without impairing the catalytic activity (potential or present). This suggests that one tryptophan residue is more accessible to oxidation than the other three, and is not essential for enzymatic activity. The manner in
METHODS FOH CLEAVAGE AND MODIFICATION OF PROTEINS
305
which enzymatic activity is lost during further oxidation (cf. Figs. 24 and 25) suggests the implication of at least one of the remaining three tryptophan residues in the maintenance of catalytic activity. The residual activity is an inherent property of the oxidized proteins and is not due to intact starting material. The following observations support this view: (a) The specific activity of various NBS treated trypsins could not be altered by fractional precipitation with ammonium sulfate. ( b ) Enzyme samples which had lost considerable activity towards BAEE were capable of incorporating 1 mole of phosphorus (from DFP) per mole of protein; this rules out unoxidiaed protein as a source for residual activity. Trypsin which had lost nearly 98% of its activity towards BAEE could still incorporate 0.61 mole of phosphorus, a value which is nearly thirty times the expected uptake if the residual 2 % activity were due to intact trypsin. (c) The DIP-derivative of oxidized trypsin (which had 50% activity prior to treatment with DFP) was found to be homogeneous in the ultracentrifuge, and to sediment at the same rate as the DIP-derivative of unoxidized trypsin. The oxidation of trypsin by NBS affects its activity towards BAEE, a typical substrate, without impairing its ability t o react with DF’P. The phosphorylat ion site which is currently considered to be a part of the “active site” of the enzyme molecule (Oosterbaan et al., 1955; Schaffer et al., 1957; Dixon et al., 1958a) apparently remains unaffected during the oxidation. This observation demonstrates the necessity of other factors besides an intact phosphorylation site for the optimal function of the enzyme. Similarly, Wood and Balls (1955) have shown that enzymatic oxidation of a single tryptophan in chymotrypsin lowered its catalytic activity to 50 % without aff ectiiig its ability to be phosphorylated. They considered tryptophan t o be an auxiliary group, necessary for full activity. The inactivation may be the result of a slight disorientation (a small increase in levorotation accompanies the oxidative inactivation of trypsin) of the active configuration of the protein molecule, brought about by the modification of one or more tryptophan residues. The actual role of tryptophan in relation to enzymatic activity remains to be elucidated. Likewise, the addition of NBS to chymotrypsin (Viswanatha and Lawson, 1961) led t o a proportional and instantaneous decrease in tryptophan absorption at 278 mp. Calculation of tryptophan from the observed maximum decrease in ahsorbance a t 278 mp gave a value of 7.36 moles of tryptophan residues per mole of protein. This value is in fair agreement with the reported tryptophan content of chymotrypsin (Weil el al., 1953; Wood and Balls, 1955). The relationship between the decrease in absorption a t 278 mp and the amount of NBS added is shown in Fig. 26. The amino acid contents of acid hydrolyzates of chymotrypsin and the oxidized chymotrypsin were the
306
B. WITKOP
same within experimental error with the exception of tyrosine. The oxidized chymotrypsin possesses one tyrosine residue less than the unoxidized material. Influence o n enzymatic activity. The effect of NBS treatment on the enzymatic activity of chymotrypsin is shown in Fig. 27. The activity diminishes rapidly with increasing addition of NBS in the initial stages, but this effect seems to become smaller with further addition of NBS. With
A TREATMENT WITH NBS
B TREATMENT WITH N B A
-
MOLES OF TRYPTOPHAN OXIDIZED
FIG. 27. Change in activity of chymotrypsin as a function of tryptophan oxidtttion. (A) Treatment with NBS; (B) treatment with NBA. From Viswanatha and Lawson (1961).
N-bromoacetamide, which is less reactive than NBS a t p H 4.0 and consequently requires more time to oxidize tryptophan residues, similar results were obtained (Fig. 27, curve B). Extrapolation of the initial steep portion of the inactivation curve obtained with NBA (Fig. 26) givesa value for tryptophan destruction of about 1 mole per mole of protein. This might suggest t,he association of one of the seven tryptophan residues present in the chymotrypsin molecule with the maintenance of catalytic activity. A similar involvement of one tryptophan residue in the reaction of diisopropylphosphorofluoridde with a-chymotrypsin is suggested by comparison of
METHODS FOR CLEAVAGE AND MODIFICATION
307
OF PROTEINS
the reactivities of the tryptophan in a-chymotrypsin with that in a-DIPchymotrypsin toward NBS (Wooton and Hess, 1960). When the precursor, chymotrypsinogen, was treated with NBS the extent of activity appearing on subsequent activation of the precursor diminished with increasing addition of the oxidant. Chymotrypsinogen treated with 10 moles of NBS per mole of protein did not give rise to active protein on activation with trypsin. Reaction with DFP. Chymotrypsin preparations oxidized with NBS to produce varying degrees of inactivation were treated with DFP and their phosphorus content then determined. An enzyme sample which had lost more than 60% of its original activity was still able to incorporate 1 mole of phosphorus (DIP-group) per mole of protein. Samples with enzymatic activity equal to 15% of the initial value or less incorporated 0.6 mole of phosphorus per mole of protein. The data on the extent of phosphorus TABLEXXVI Phosphorus Content of Oxidized Chymotrypsin ( C H T ) Samples Reacted with DFPa No.
Sample
1 2
Chymotrypsin Oxidized CHT Oxidized CHT Oxidized CHT Oxidized CHT
3 4 5
Moles NBS Mole phosper mole Per cent activity phorus per CHT mole protein
0 2
100
4
38
1.01 1.37 1.13
10 10
15 10
0.62 0.58
68
Viswanatha and Lawson (1961).
incorporation into various chymotrypsin samples during reaction with DFP is given in Table XXVI. The low phosphorus value in the highly oxidized chymotrypsin samples treated with DFP might be related to the protein denaturation, a secondary phenomenon that accompanies the action of NBS. The results obtained with DFP are in agreement with the earlier report of Wood and Balls (1955), who showed that enzymatically oxidized a-chymotrypsin with 50 % of the initial activity could still react with DFP. Reaction of N P A . Chymotrypsin samples oxidized with NBS to different degrees of inactivation were treated with NPA or C14-labeled NPA at pH 5.0 or at pH 6.0. The acetylation of the enzyme by NPA is retarded by the NBS oxidation of the protein. The rates of acetylation of various oxidized chymotrypsin samples at pH 5.0 paralleled their relative activities toward N-acetyl-L-tyrosine-Ethyl ester (ATE). However, the net release of p-nitrophenol, measured after the rate of its liberation had approached that of spontaneous hydrolysis, corresponded to a considerable acetylation
308
B . WITKQP
of the protein even though catalytic activity towards ATEE waslow. Thus, oxidized chymotrypsin which possessed only 6 3‘ 4 of its original activity toward ATEE, yielded 0.54 mole of p-nitrophenol on treatment with S P A at pH 5.0. The results arc summarized in Table XXVII. TABLEXXVII Data Sunimarizing the Reaction of Oxidized Chyniotr ypsin Samples with N P A at p H 6.0“ Sample No. 1
2 3 4 5
Moles NBS per mole CHT 0 2 3 4 7
Activity toward ATEE 100 40 30 20
0.0
Rate of “burst”*
Per cent “burst”c
Relative “tnirst”‘l
100 42 35 21 11
83 74 63 50 45
1.o 0.89 0.76 0.60 0.54
Viswanatha and Lawson (1961). Iriitiul rate relative t o sample 1. c Per cent of theoretically expected value. d In the case of highly oxidized samples the value is :L innximum (1
TABLE XXVIII Data Showing the Zncorporation of CL4-Acetate b y Oxidized Chyniotrypsin Samples during the Reaction with CI4-Labeled NPA.3 6 No.
CHT Oxidized Oxidized Oxidized Oxidized Oxidized
1 2 3 4 5 6 ~~
~
CHT CHT CHT CHT CHT
0 2 4
6 8 10
100 60.6 48.5 27.3 18.2 9.1
148,000 120,000 128,000 107,000 89,000
77,000
1.18 0.8 1.03 0.813 0.71 0.62 ~
Viswanatha and Lawson (1961). b Chymotrypsin reacted with CL4-NPA at p H 5.0 and acetyl enzyme prepared as described in the text. The Cl4-content of acetyl enzymes was measured. One pniole of NPA corresponds t o 125,000 cpm. The amount of acetate combined with t h e enzyme is calculated by dividing t h e CI4-contentper pmole protein by 125,000. a
Similar results were obtained when oxidized chymotrypsin samples were treated with CI4-labeled NPA a t pH 5.0 for 1 hr and the C14-content of the isolated acetyl enzymes were measured. Thr data ohtnincd are given in Table XXVIIT.
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
309
When the reaction with NPA was carried out at pH 8.0, the nitrophenol liberation due to the fast acetylation reaction was similar to those obtained in the experiments of p H 5.0. The catalysis of NPA hydrolysis subsequent to the initial fast reaction paralleled the activity towards ATEE. The C14-labeled acetyl derivatives of various oxidized chymotrypsin samples were allowed to deacetylate at pH 8.0. It was found that the oxidized enzymes lost the acetate much slower than the unoxidized enzyme.
FIG.28. Rate of deacetylation of C14-labeled acetylchymotrypsin (ACHT). (A) Acetylchymotrypsin prepared from NBS-oxidized enzyme with 47% of original activity; (€3) acetylchymotrypsin prepared from NBS-oxidized enzyme with 10% activity; (C) acetylchymotrypsin from unoxidized enzyme. 0, buffer pH 8.0; A,buffer pH 8.0 containing ATEE (fifty-fold molar excess of ATEE over the enzyme). The Cl4-content of the various trichloroacetic acid (TCA) precipitates appears to be a true measure of the deacetylation process prior to the addition of TCA. Further dialysis of acid solutions of TCA precipitates against dilute HCI did not lead to any significant change in the C14-activity. (D) Appearance of enzyme activity. Acetylchymotrypsin added to a solution of ATEE of pH 8.0 (the figure shown is traced from the actual record of the Cary spectrophotometer). ATEE, 2 X 10-8 M ; ACHT, 10 p g ; pH 8.0. From Viswanatha and Lawson (1961).
The greater the degree of NBS oxidation of the enzyme the slower was the rate with which its acetyl derivative parted with the acetate residue. The results are shown in Fig. 28. Interestingly enough the acetyl derivative of unoxidized chymotrypsin lost its acetate residue at a much slower rate than the rate of appearance of maximum enzymatic activity. The half-ttime of deacetylation was about 3 min, whereas maximal activity with t,he acetylchymotrypsin was obtained about 1 .O-1.5 min after its addition to substrate at pH 8.0. The modification brought about by NBS oxidation seems to interfere
3 10
B. WITKOP
with both the acylation and the deacylation steps without changing the K , value for the substrate. It is interesting to contrast this oxidative inactivation of chymotrypsin with that caused by reaction with dinitrofluorobenzene (Massey and Hartley, 1956), in which the inactivation was accompanied by an increase in K , value, while K S remained constant. Another interesting observation, not related to NBS oxidation, is the rate of deacetylation of acetylchymotrypsin compared to the rate of appearance of activity. No direct measurements of the deacetylation of acetyl enzyme has heretofore been reported. The use of CI4-acetatelabeled NPA has made this study possible and the results indicate that the acetate is lost at a much slower rate than the rate of appearance of enzyme activity. The apparent discrepancy in the rates of deacetylation obtained by direct measurement in this study with those of indirect measurements (Dixon and Neurath, 1957) remains to be resolved.
VI. INTRODUCTION OR PREVENTION OF ENZYMATIC CLEAVAGE BY CHEMCIAL MODIFICATION Chemical and enzymatic methods for the selective cleavage of peptide bonds are not completely separate either-or propositions. In addition chemical modification may well be combined with enzymatic degradation in a manner which promises new usages and more selectivity for proteinases and peptidases in sequence studies. The cleavage of a protein by trypsin may be selectively restricted to the arginine peptide bonds by blocking the s-NH2 groups of the lysine residues by conversion to 6-N-carbobenzyloxygroups (Anfinsen et al., 1956). Fewer and larger peptides are then obtained by tryptic digestion of the modified protein. A modification of this procedure is the use of dinitrofluorobenzene which produces 6-DNP-lysine derivatives. A disadvantage of both methods is the loss of the basicity and of the hydrophilic character of the e-NH2 lysine groups. This may be prevented by guanidation of the protein. In this process the free and accessible lysine eNH2 groups are converted to guanido groups. The resulting homoarginine peptide bonds are largely untouched by trypsin (Weil and Telka, 1957). groups of A modification of this approach is the conversion of the E-NH~ lysine into dithiocarbamate derivatives by reaction with carbon disulfide at alkaline pH. Figure 29 shows the results of the tryptic digestion of the 20-residue S-peptide before treatment with CSZ, after treatment with CS2, and repetition of the enzymatic cleavage after easy removal of CSz with liberation of the originally blocked c-NH2 group of lysine (Merigan et al., personal communication). The removal of arginine from attack by trypsin, in principle, is possible through the use of sodium in liquid ammonia. The result is a protein con-
I*
I*
rn
r
Ribonuclease S -peptide structure
2 b M
Products of tryptic digestion of CS, modified S-peptide
[Lys-Glu-Thr
Products obtained after repeated tryptic digestion (following CS, removal)
(Lys-Glu-Thr-A!a-Ala-Ah-Lys~ ~~
I
-Ala-Ala-Ala-Lys-Phe-Glu-Argl
~
1-
I
I(Glu, His, Met, Asp, Ser,, Thr, Ala,)l
](Glu, His,Met, Asp, d r , ,Thr,,Ala,)l
* y
g
~
*Bonds which are susceptible to tryptic digestion i n the unmodified molecule. FIG.29. Tryptic digestion with and without CSz modification. T. Merigan, W.J. Dreyer, and A. Berger (personal communication).
v
TIe
m
312
R . WITKOP
taining ornithine instead of arginine. The ornithine peptide bond is stable t,o the action of trypsin. The method has been used for the “deguanidation” of gelatin (Berger et al., 1958). The deguanidation under conditions of electrolysis has been noted previously (Stein and Moore, 1961). The opposite approach, namely, the creation of additional lysine-like sites for cleavage by trypsin has been used in the conversion of cysteine bonds to S-(6-aminoethy1)-cysteine residues by reaction of the thiol groups with 6-bromoethylamine (Lindley, 1959). Enzymatic degradation of such modified proteins, e.g., reduced and p-aminoethylated insulin, by the acTABLEXXIX Dibasic Esters with Length of Chain and Separation of Cationic Charges, Comparable to Ethyl Lysinate, Which Serve as Substrates for Trypsina Formula e
b
&N-CH
2-CH 2-CH 2-CH 2-CH (NHs)-COOEt
Ethyl lysinate
b
H1N-CHz-CH
2-CO-NH-CH2-COOEt
Ethyl-6-alanylglycinnte
2-CHr-CH
Ethyl-t-aminocaproate
@
H3N-CHz-CH
2-CH z-COOE t
b
H3N-CHz-CO4-CH
z-CH-COOEt
I
w
NH-COCHz-NH3
Ethyl-N ,O-diglycyl-nr,serinate
m
H3fi-CH z-CO--O-CH
2-CH-COOEt
I
NH-COCaH6 0
Ethyl-N-benzoyl-O-glycyl-nL-serinate
Shalitin (1961)
tion of trypsin and carboxypeptidase B (Tietze et nl., 1957) has been ohserved. Conversion of the free y-glutamyl carboxy groups to y-hydraxides makes peptide bonds in model compounds (and probably in proteins) next t,o these modified glutamic acid residues susceptible to splitting by trypsin (Ebata and Akabori, 1960; Ebata, 1961). Extensive modifications of the lysine chain do not significantly affect the ability of the analogs to serve as substrates for tryspin. The compounds in Table XXIX were hydrolyzed by trypsin a t rates comparable to that of ethyl lysinate. The introduction of glycyl residues into proteins with the Leuchs anhydrides of glycine makes peptide bonds next to serine susceptible to cleavage by trypsin (Shalitin, 1961).
METHODS FOR CLEAVAGE AND MODIFICATION OF PEOTEINS
313
VII. CONCLUSION There is a growing conviction among organic chemists that the scientific frontier of organic chemistry has moved from small molecules to the large entities that are involved in the process of the formation and preservation of the living cell. Consequently the skills of the organic chemist wisely should be applied to the design of special methods for the analysis, degradation, and synthesis of large molecules. A comparative macromolecular methodology will be written some day and will demonstrate the simplicity of the underlying concepts. Certainly, all the reactions utilized for the selective cleavage of proteins are conceptually not new and, in retrospect, surprisingly simple. This preview may serve a useful purpose in delineating, perhaps for the first time, the criteria and requirements that are essential to selective cleavage. In principle, all selective cleavages are, or must be, variations of a key theme,
OPO,H 0I ..... ..
'
+ EUI/- EtI
Liberated guanine
OP0,H
o.... l
I
Bound guanine in DNA
I bP02H O..--.I "Deguanidated" DNA
314
B. WITKOP
namely, intramolecular acceleration of displacement or elimination reactions to the point where they will occur under extremely mild conditions. The formulation and recognition of this principle, so fruitful for protein chemistry, may stimulate and influence the search for better methods in neighboring fields. Sometimes the same agent may serve many purposes. As an example may be cited the principle of alkylation and subsequent cleavage which was used for selective splitting of methionine-containing proteins. An analogous reaction has led to the “depurination” of deoxyribonucleic acid (DNA) by selective N-alkylation of guanine residues (Bautz and Freese, 1960). Another example is the selective oxidation by periodic acid which is used in the breakdown of proteins (Gross and Witkop, 1960), polysaccharides (Barry, 1943), and nucleic acids (Whitfield, 1954; Brown, et al., 1955; cf. Reese et al., 1960). The rate of reaction of the oxidant with the reactive products and their utilization for further displacement and elimination reactions are the variables which, when properly manipulated, will make for useful cleavage methods applicable to all three kinds of macromolecules. ACKNOWLEDGMENTS I am greatly obligated to my colleagues and associates at the National Institutes of Health who contributed experimentally, conceptually, and editorially to this chapter. Special thanks are due to Drs. L. A. Cohen, E. Gross, and W. B. Lawson. Dr. C. B. Anfinsen, National Heart Institute, and Drs. A. Berger, A. Patchornik, and M. Sela from the Weiamann Institute were extremely helpful in making unpublished material available prior t o publication. T o them and many other cooperative authors I extend my sincere thanks.
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Shalitin, Y. (1961). Abstr. Proc. 4th Congr. Sci. SOC.Bull. Research Council Israel 26, 65. Shapiro, S. K., and Mather, A. N. (1958). Arch. Biocheni. Biophys. 238, 631. Siehr, D. J. (1961). J . Am. Chem. Soc. 89, 2401. Slobodian, E., and Levy, M. (1953). J . Biol. Chem. 201,371. Snyder, H. R. (1942). J . Am. Chern. SOC.64,2082. Spackman, D. H . , Stein, W. H., and Moore, S. (1958). Anal. Chem. 30, 1190. Spackman, D. H., Stein, W. H., and Moore, S. (1960). J . BioE. Chem. 286, 656. Springell, P. H. (1961). Nature 191, 1372. Stein, W. H . , and Moore, S. (1946). J . Org. Chem. 11,681. Stein, W. H., andMoore, S. (1951). J . Biol. Chem. 190, 103. Stewart, J. A., and Ouellet, L. (1959). Can. J . Chem. 37,751. Stieglitz, J. (1896). Am. Chem. J . 18,755. Stirling, C. J. M. (1960). J . Chem. SOC.p. 255. Swan, J. M. (1958). In “Current Trends in Heterocyclic Chemistry” (A. Albert, G. M. Badger, and C. W. Shoppee, eds.), p. 65. Academic Press, New York. Synge, R. L. M. (1943). Chem. Revs. 32, 135. Synge, R. L. M. (1949). Biochem. J . 44, 542. Thompson, E. 0. P. (1960a). In “Advances in Organic Chemistry” (R. A. Raphael, E. C. Taylor, and H. Wynberg, eds.) Vol. I, p. 149. Interscience, New York. Thompson, E. 0. P. (1960b). Australian J . Biol. Sci. 13, 106. Tietze, F., Gladner, J. A., and Folk, J. E. (1957). Biochim. et Biophys. Acta 26, 659. Toennies, G., and Kolb, J. J. (1945a). J. Am. Chem. SOC.67, 849. Toennies, G., and Kolb, J. J. (1945b). J . Am. Chem. Soc. 67, 1141. TomBBek, V., HoleyBovskf, V., Mikeb, O., and gorm, F. (1960). Biochim. et Biophys. Acta 38, 570. Tristram, G. R. (1953). I n “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. IA, p. 215. Academic Press, New York. Tsugita, A., Gish, D. T., Young, J., Fraenkel-Conrat, H., Knight, C. A,, and Stanley, W. M. (1960). Proc. Natl. Acad. Sci. U . S. 46, 1463. Tuppy, H. (1953). Biochim. et Biophys. Acta 11, 449. Tuppy, H., and Bodo, G. (1954). Monatsh. Chem. 86,1182. Tuppy, H., and Michl, H. (1953). Monatsh. Chem. 84,1011. Tuppy, H., and PalBus, S. (1955). Acta Chem. Scand. 9, 353. Vajda, T. (1959a). Chem. & Ind. (London) p. 197. Vajda, T. (1959b). Acta Chim. Acad. Sci. Hung. 21, 71. Vanderhaeghe, H., and Parmentier, G. (1961). J . Am. Chem. SOC.82,4414. Viswanatha, T. (1957). Compt. rend trau. lab. Carlsberg Skr. chim. 13, 183. Viswanatha, T., and Irreverre, F. (1960). Biochim. et Biophys. Acta 40,564. Viswanatha, T., and Lawson, W. B. (1961). Arch. Biochem. Biophys. 98, 128. Viswanatha, T., Lawson, W. B., and Witkop, B. (1960). Biochim. et Biophys. Aclo 40, 216. Vithayathil, P. J., and Richards, F. M. (1960). J . B i d . Chem. 236, 2343. vonBraun, J., andEngelbertz, P. (1923). Ber. 66,1573. von Braun, J., and Murjahn, R. (1926). Bey. 69,1202. von Braun, J., May, W., and Michaelis, R. (1931). Ann. 490,189. Weil, L., and Telka, M. (1957). Arch. Biochem. Biophys. 71,473. Weil, L., James, S., and Buchert, A. R. (1953). Arch. Biochem. Biophys. 46,266. White, E. H. and Schemer, H. (1961). Abstr. Am. Chem. SOC.Meeting, Chicago, September, 1961 p. 2Q. White, F. H., Jr. (1960). J . B i d . Chem. 236,383.
METHODS FOR CLEAVAGE AND MODIFICATION OF PROTEINS
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Whitfield, P. R. (1954). Biochem. J . 68, 390. Wieland, Th., and Pfleiderer, G. (1957). I n “Advances in Enzymology” (F. F. Nord, ed.), Vol. XIX, p. 262. Interscience, New York. Wieland, Th., and Schopf, A. (1959). Ann. 626, 174. Wieland, Th., Shin, K. H., and Heinke, B. (1958). Chem. Ber. 91, 483. Witkop, B. (1956). Ezperientia 12, 372. Witkop, B. (1960). Abstr. 7th Natl. Med. Chem. Symposium, Am. Chem. Soc., Rhode Island, June, pp. 9a-9e. Witkop, B. (1961). Tampaku-Shitsu, kaku-san, k6sd (Proteins, Nucleic Acids, Enzymes) 6, 557 (in Japanese). Witkop, B., and Beiler, Th. (1956). J . A m . Chem. Soc. 78. 2882. Wittmann, H. G., and Braunitzer, G. (1959). ViroZogy 9,726. Wood, H. N., and Balls, A. K. (1955). J . BioE. Chem. 213, 297. Wooton, J. F., and Hess, G. P. (1960). Nature 188, 726. Yaron, A . , Patchornik, A . , and Sela, M. (1961). Personal communication. Zahn, H., and Traumann, K. (1954). 2.Naturforsch. Pt. QB,518. Zahn, H., and Ziirn, L. (1956). Ann. 613,76. Zahn, H., and Ziirn, L. (1958). Chem. Rer. 91, 1359. Zervas, I,.,Winitz, M., and Greenstein, J. P. (1961). J . A m . Chem. Soc. 83, 3300. Ziirn, L. (1960). Ann. 631,56.
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THE VISCOSITY OF MACROMOLECULES IN RELATION TO MOLECULAR CONFORMATION By JEN TSI YANG Cardiovascular Research Institute and Department of Biochemistry, University o f California Medical Center, San Francisco, California'
I. Introduction.. ................ ...................... 323 11. Definitions and Equations, .... ...................... 326 A. Definitions . . . . . . . . . . . . . . . . . ............................ 326 B. Determination of Intrinsic Viscosity. . . . . . . . . . . . . . . . . . .......... 328 C. Intrinsic Viscosity-Molecular Weight Relationshi . . . . . . . . . . . . . . . . . . 329 .................. 331 111. Newtonian Viscosities.. ............................ A. Theories for Rigid Particles .................... B. Determination of Particle Shape from Intrinsic Vi C. Other Hydrodynamic Properties. . . . . . . . . . . . . . . . . . D. Concept of Equivalent Hydrodynamic Ellipsoid.. E. Estimation of the Length of Equivalent Ellipsoid. F. Theories for Flexible Coils.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 G. Polyelectrolytes and Electroviscous Effect . . . . . . . . ............. 349 H. Examples of Several Common Proteins and Polype IV. Non-Newtonian Viscosities.. ............................................ 363 A. Theories for Rigid Particles. ........................................ 363 B. Determination of Particle Length (or Diameter). . . .............. 364 C. Experimental Confirmation, ......................................... 365 D. Comparison with Flow Birefringence Method.. ....................... 368 E . Theories for Flexible Coils.. ... ...................... F. Power Law of Viscosity.. . . . . . . ................... G. Complex Viscosity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Experimental Methods ........................................ 375 A. Types of Viscometers.. . . . . . . . . . . . . . . . . . . B. Corrections for a Capillary Viscometer . . C. Extrapolation to Zero Rate of Shear.. . . . . . . . . . . . . . . . . . . . . . . . . . 3% D. Design of a Capillary Viscometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 E . Viscosities of Extremely Dilute Solutions .................... 387 VI. Conclusions., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Appendix (Tables VIII-XIII) . . . . . . . . . . . . . . . References. .................................
I. INTRODUCTION Of the quantities which characterize a polymer solution the viscosity is one of the simplest and cheapest to measure; for this reason it is also among 1 This work was begun at and initially supported by the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire.
323
324
J E N TSI YANG
the most intensively studied, theoretically as well as experimentally. Thc importance of viscometry in industrial applications is reflected by the rapid progress of the science of flow and deformation-rheology. To protein chemists the determination of intrinsic viscosity (Section 11, A ), has become so fundamental that rarely is the size and shape of a protein in solutions characterized without a t least including a set of viscosity data. The specific excuses for the present review are threefold: first, the interpretations of intrinsic viscosity of protein solutions has recently undergone a more careful reevaluation. There have arisen doubts concerning aspects of viscosity treatments routinely applied in characterizing proteins, particularly the asymmetry of the protein molecules. These doubts must be examined and, if possible, resolved; the proper applications and limitations of viscometry in the studies of proteins will be emphasized. Second, the recent development of the theories of non-Newtonian viscosity has made the gradient dependence of viscosity less of a nuisance and more a source of additional information concerning the shape of polymers. It also offers a new means for studying the conformations and conformational changes of proteins. It is therefore opportune to put this new technique in the hands of biochemists, although little experimental work has as yet been done. Third, it appears desirable to summarize the basic equations employed in viscosity measurements, and the complications that may arise and precautions that are required under normal experimental conditions. No attempt will be made in t,his review to describe all the theoretical t,reat,ments even in a cursory manner. Only the physical significance of several well-known equations will be quoted without derivations. This it is hoped should be adequate for our purpose. A good “feel” for the relation between viscosity and macromolecular structure may be gained by perusing Table I. Globular proteins generally have lower intrinsic viscosities than flexible polymers (e.g., polystyrene) of comparable molecular weight; it is to be expected that compact, rigid proteins will move through solvent easier than “swollen” chains. There is however, no correlation between intrinsic viscosity and molecular weight when proteins of different types are considered; the intrinsic viscosity of rigid particles is dominated by particle asymmetry, not size. Thus, for example, in spite of its high molecular weight tobacco mosaic virus sohtions have a much smaller intrinsic viscosity than collagen solutions. On the other hand, in homologous series of one molecular type, rigid (e.g., a-helical poly-7-benzyl-L-glutamates) or flexible (e.g. , polystyrenes), intrinsic viscosities do increase with molecular weights. This is attributed to an increase in asymmetry (for rigid particles) or in effective volume (for flexible polymers) with increasing molecular weights. (Idealized hard spheres are an exception; see Section 111, A . ) The intrinsic viscosity of a
32,s
VISCOSITY OF MACROMOLECULES
flexible polymer is strikingly dependent on the medium in which it dissolves; it is always greater in a "good" solvent (high polymer-solvent affinity) than in a "poor" one (low polymer-solvent affinity), again due to the difference in effective volumes. Among tlhe flexible polymers the TABLEI Intrinsic Viscosities of Several Proteins and Polymers i n Solutionsa Substance
Molecular weight
Proteins in aqueous solutions a t isoelectric points Ribonucleaseb Myoglobinc Ovalbumind Serum albumin6 Hemoglobin' Fibrinogen# Collagenh Myosin i Tobacco mosaic virusi Poly -r-beneyl-L-glutamatesb (1) I n dimethyl formamide (2) In dichloroacetic acid at 25°C
Polystyrenes (1) In benzene at 20"CE (2) In 0.869:0.131 (v/v) cyclohexane/carbon tetrachloride at 15"Cm Cellulose trinitrate in acetone a t 25"Cn a For the definition of intrinsic viscosity and related terms, see Table 11. Buzzell and Tanford (1956). c Wyman and Ingalls (1943). d Polson (1939). "Loeb and Seheraga (1956); Champagne (1957). f Tanford (1957b). g Scheraga and Laskowski (1957).
13,700 17,000 44 000 66 ,000 67,000 335,000 345,000 493,000 39,000,000
[s] dl gm-1
0.033 0.031 0.043 0.039 0.036 0.25 11.5 2.17 0.37
66 ,500 340 ,000 66,500 340,000
0.45 7.20 0.45 1.84
65,500 262,000 2,550,000 3 220,000
0.36 1.07 5.54 1.35
77 000 400,000
1.23 6.50
Boedtker and Doty (1956). Holtzer and Lowey (1956, 1959). j Boedtker and Simmons (1958). k Doty et aE. (1956). (1) a-helicnl form; (2) randomly coiled form. 2 Fox and Flory (1951). I'assaglia et al. (1960). n Holtzer et al. (1954). h
i
intrinsic viscosity also depends, as expected, on structure, being greater the more extended the polymer chains (cf., cellulose trinitrate and polystyrene). The foregoing observations merely suggest, the many complexities of the viscosity of macromolecules in solutions and emphasize the need for co-
326
JEN TSI YANG
ordinating viscometry with other physicochemical methods. For instance, viscosity measurements alone cannot distinguish the contributions due to the asymmetry of the protein molecule from the effective volume of the molecule in solution. Indeed there are many assumptions involved in the interpretation of intrinsic viscosities of protein solutions which will be discussed in more detail in Section 111. Intelligently used, however, viscometry can be a surprisingly versatile method. One of the most fruitful approaches has been the study of conformational changes of the protein molecules in solutions. The viscosity measurements have been used to detect the ill-defined process “denaturation,” to follow its kinetics, and sometimes even to establish its reversal. But even here success must be tempered with caution, for had such studies been made with only one poly-y-benzyl-L-glutamate having a molecular weight of about 66,500 (Table I) they would have failed ignominously. Likewise the increase of intrinsic viscosity on denaturation of a globular protein and its decrease on reversal of denaturation would not be sufficient enough to demonstrate a direct reversible process. But viscometry can provide powerful evidence when taken in conjunction with other measurements such as optical rotatory power and deuterium-hydrogen exchange. Today protein chemists are no longer preoccupied with the elucidation of the bare outlines (size and shape) of the protein molecules and are beginning to probe deeply into the internal structures of these molecules. It is in this realm that viscometry along with other methods will find new importance in the studies of conformations and conformational changes of the proteins.
11. DEFINITIONS AND EQUATIONS
A . Definitions By Newton’s definition the viscosity or, more appropriately, the viscosity coefficient, q , of a fluid in a laminar steady-state flow is expressed as the tangential force, F , per unit area, A , required to maintain a unit rate of shear (or velocity gradient), G, in the liquid. If the liquid fills the space between two parallel planes of area, A , one of which moves at a constant distance, r, from the other with a relative velocity, u, then we have Shearing stress: r = F / A Rate of shear: Viscosity:
in dynes cm-2
G = du/dr in sec-‘ 11 = r / G
in dynes sec cm-’
(14
(1b) (1c)
The viscosity is said to be Newtonian, if it is independent of the applied shearing stress; and non-Newtonian, when it does not conform to New-
327
VISCOSITY OF MACROMOLECULES
ton’s original definition. Some workers, however, have also suggested the term “generalized Newtonian viscosity” instead of using the prefix “non.” The viscosity as defined in Eq. ( l c ) may also be considered as a measure of the energy dissipation in the fluid required to maintain a viscous flow. The rate of work, dW/dt, done in overcoming the frictional resistance in a volume, V , of the liquid is equal to G2q per unit volume (Robinson, TABLEI1 Definitions and Symbols of Several Viscosity Terms Definition
VIP
who
Conventional Symbol
Name Solution viscosity Solvent viscosity Kinematic viscosity Relative viscosity Specific viscosity Reduced viscosity Inherent viscosity Intrinsic0 viscosity
Proposed Symbol
Name
Viscosity of the solution lo Viscosity of the pure solvent q/p Viscosity/density ratio )I/)I~Solution/solvent ratio or viscosity ratio 9
-
Viscosity number
[)I]
Logarithmic viscosity number Limiting viscosity numberb
First suggested by Kraemer (1938) (see also, Kraemer and Lansing, 1935). Concentration unit: grams per 100 ml (or grams per deciliter). More recently, also designated as the “Staudinger Index” by the International Union of Pure and Applied Chemistry (1957). Concentration unit: grams per milliliter.
1939). The introduction of macromolecules into the fluid disturbs the streamlines, thus causing an additional energy dissipation, the amount of which depends upon the size and shape of the particles. As a result the viscosity of a polymer solution is always greater than that of the pure solvent. The conventional names and symbols of viscosity of solutions and also the proposed terminology as approved by the International Union of Pure and Applied Chemistry (1952) are listed in Table 11. Logically speaking, the designation of [v], vre1 , and vsp as “viscosities” is indeed incorrect since these quantities do not have the dimensions of
328
J EN TSI YANG
viscosity. The proposed new terminology, however, appears not to have been accepted universally, perhaps partly because of tradition and partly because some of the terms are rather clumsy in practice. It is noted that the concentration units in the two systems differ by a factor of 100. For example, the intrinsic viscosity of serum albumin is 0.040 dl gm-' or 4.0 ml gm-'. Unfortunately this has caused some confusion in the recent literature because many publications, both experimental and theoretical, still prefer the old expressions. Without an explicit statement of the units employed, misunderstanding often occurs. In this review the old conventions are used, mainly because the majority of publications on proteins appear not to have adopted the new symbols.
B. Determination of Intrinsic Viscosity Xumerous empirical equations have been proposed to express the viscosities as a functioii of concentrations for the determination of intrinsic viscosity. These equations have been tabulated in several textbooks (see, for example, Philippoff, 1942), but many of them have only limited applications and are already obsolete. Here we will only mention three commonly used ones. For dilute solutions the well-known Huggins equation (1942) can be written as
[?I
+ k"rl12C
(2a) where the coefficient k' is believed to be some function of solute-solvent interaction. Another form which is related to Eq. (2a) has also been used extensively (Kraemer, 1938) : %P/C
=
(loge B r e l ) / C
= [B]
-~"[BI~C
(2b)
By expanding (log, qr,l)/C into a power series of ?lSp/C",as the conccntration approaches zero, and combining them with Eq. (2a) we have
k'
+ k"
= 0.5
'l'hus by plotting both qs,/C and (log, vre~)/Cagainst the concentration according to Eys. (2a) and (2b) one should obtain the same intercept, [?I, and the limiting slopes at C = 0 should satisfy the above condition for k' and Id'. This double check would enable us to determine the intrinsic viscosity with more confidence. Another useful equation for moderately concentrated solutions (up to 5 % or more) was proposed by Martin ( 1942) (see also Spurlin et al., 1946) ; log10
(%P/C)
=
log10 [a1
+ I'C[rllC
which at low concentrations is related to the Huggins equation with lc = lc'/2.303
(3)
VISCOSITY OF MACROMOLECULES
329
Martin's equation may find applications in the cases where proteins denature readily at interfaces, especially when flowing through a capillary a t high dilutions. It has been used also in the study of non-Newtonian viscosities (see Section IV). However, in a so-called good solvent, i.e., where strong solute-solvent interaction exists, the Martin plot [log,, ( T*,/C) versus C ] frequently shows a downward curvature as the concentration decreases below a certain range (Weissberg et al., 1951). As a consequence one may obtain a false intrinsic viscosity if the experimental data are limited only to the range of high concentrations. For solutions of high concentrations, Raker ( 1913) has suggested the following equation : ?re1
=
+ ac)"
(4a)
+ [olC/n)"
(4b)
(1
which can he rewritten as orel
= (1
where a and n are two constants. It reduces to the Huggins equation as the concentration approaches zero. On the other hand, it converts into the Arrhenius equation when n becomes infinite, i.e., qrel = e"'" (or log, vrel = [qJC). Equation (4) may be of limited use for routine viscosity measurements of protein solutions, but Oncley et al. (1947) have tried the Arrhenius equation for very concentrated protein solutions in an effort to avoid the complication of surface denaturation. It is pertinent to note that all these empirical equations are useful only in certain limited ranges of concentrations and therefore one should be careful in applying them to experimental results, It is always a good practice to design the experiments so that Eq. (2a) can be applied, unless other considerations prevent its use.
C. Intrinsic Viscosity-MolecularWeight Relationship The dependence of the intrinsic viscosity on the molecular weight for a homologous series has customarily been expressed by the modified Staudinger equation, often called the Mark-Houwink equation (Mark, 19%; Houwink, 1940) :
[v]
=
KM"
(5)
where K and a are two constants for a given solute-solvent system, the latter being characteristic of the molecular conformation. For rigid spheres a = 0 and for rigid ellipsoids and rods a = 1.7-2, whereas for flexible polymers it varies from 0.5 to about 0.8. It has further been observed experimentally that stiff polymers such as cellulose derivatives and nucleic acids have values for a ranging from 0.8 to 1.2. Staudinger was among the first to emphasize the importance of viscosity measurements for molecular
330
JEN TSI YANG
weight determinations of high polymers and to arrive empirically at a value of unity for the exponent for certain systems studied (Staudinger, 1932). If the polymer is polydisperse, the intrinsic viscosity is obviously related to some kind of average molecular weight, appropriately called the viscosity average by Flory (1943), which depends on the magnitude of a. It is a common practice to define the different averages of molecular weight by the general relationship:
A ? = ( Zr M, ,F / Z c,MP-l) = ( CN , M q + ' / C N , M f ) a
(6)
where c , is the weight concentration, N i the number of moles, and M , the molecular weight, of species i. Thus, for number average, M , , for weight average, M , , for z-average, M , , for ( z 1 ) = average, M , + l ,
+
a = o
a = l a = 2 a = 3
and so on. The viscosity average, M , , however, obeys a different relationship. At infinite dilution ?JSP
=
C(%*>i
and
Then we have according to Eq. (5)
M,
=
(cc i M i " / C c;)''"
=
( CN i M ; + " / C NiMi)""
(8)
Thus, for a = 1, M , is identical with M , . For a values other than unity M , has usually been found to be closer to M , than to M , , although quantitatively it depends upon the molecular weight distribution in each particular case. For example, for polymers having the "most probable distribution" (Flory, 1953)
+ a > r ( i + ~)]"":2 where r ( 1 + a) is the gamma function of (1 + a ) . Thus the ratios beM,:M,:M,
=
i:[(i
come 1:1.76:2 for a = 0.5, 1:2:2 for a = 1, and 1:2.31:2 for a = 1.7. As another illustration consider a protein containing 10 % end-to-end dimer, which gives the ratios M,: M,: M , = 1.05: 1.13: 1.10. (The exponent of 1.7 for rigid ellipsoids assumes that the components have the same minor axis and their molecular weights are proportional to the axial ratios. ItJ can be shown that the intrinsic viscosity will actually decrease if the particles form side-by-side aggregates.)
VISCOSITY OF MACROMOLECULES
33 1
111. NEWTONIAN VISCOSITIES A . Theoriesfor Rigid Particles The additional average rate of energy of dissipation per unit volume as caused by the presence of a solute or suspended particle over that by solvent in a fluid can be expressed as
(dW/dt)/V = G 2 ( q -
qo) =
G’qonvv/V
(9a)
which in turn becomes (q
-
~O>/VO
=
nuv/V =
VCP
(9b)
Here n is the number of the noninteracting, identical particles, v the volume of each particle, V the volume of the solution or suspension, @ the volume fraction of the solution occupied by the particles, and v may be regarded as the viscosity increment which depends on the shape of the rigid particles. Einstein (1906a, 1911) was the first to treat thoroughly the viscosity of a solution or suspension of spherical particles which are rigid and large relative to the size of the solvent molecules, the latter being considered in effect as a continuous medium. In other words, the particles are regarded as small enough to exhibit Brownian motion, but large enough to obey the laws of macroscopic hydrodynamics. By straightforward hydrodynamic reasoning he obtained a value of 2.5 for v in Eq. (9b) in the limiting case where @ approaches zero. It is noted that the absolute size of the spheres does not enter into the viscosity equation. For solutions of nonspherical particles the situation is more complicated and the physical picture can be described qualitatively as follows: for a system of particles in a fluid one can define a distribution function, F (Peterlin, 1938), which specifies the relative number of particles with their axes pointed in a particular direction. Under the influence of an applied shearing stress a gradient of the distribution function, aF/at, is set up and the particles tend to rotate at rates which depend upon their orientation, so that they remain longer with their major axes in position parallel to the flow than perpendicular to it. This preferred orientation is however opposed by the rotary Brownian motion of the particles which tends to level out the distribution or orientations and lead the particles back toward a more random distribution. The intensity of the Brownian motion can be characterized by a rotary diffusion coefficient 8. Mathematically one can write for a laminar, steady-state flow: aF/dt
=
8V2F - V . ( F w ) = 0
(10)
where V 2is the Laplacian operator, the operator V . denotes the divergence w is the angular velocity of the rotating par-
of the function ( F a ) , and
332
JEN TSI YANG
ticles. The function F is further characterized by two parameters, a and R. Here a is the ratio of the rate of shear to the rotary diffusion coefficient, i.e., G / 0 . According to Jeffery (1922-1923) R = ( p z - l ) / ( p 2 I ) for ellipsoids of revolution having an axial ratio of p . Thus the fundamental problem becomes to find a general solution for the distribution function I" in Eq. (10), (in terms of a and p ) which determines the distortion of the streaming lines that contributes to the increase in viscosity of the solution over the solvent. Simha (1940) employing Eq. ( 10) solved the equation for the viscosities of solutions of ellipsoids of revolution for the limiting case a -+ 0. Under this condition the distribution of the particles can be regarded as almost completely random. Simha also found that for large axial ratios the viscosity increment a t a -+ 0 for very dilute solutions or suspensions (i.e., Q, 4 0) can be approximately represented by
+
Y
= p2/15(10g, 2 p - 1.5)
+ p2/5(10ge2 p - 0.5) + 14/15
(lla)
for prolate ellipsoids and Y =
16q/15 arctan (in radians) y
( 1 lb )
for oblate ellipsoids. Here p = a / b arid q = l / p , a and b being the semimajor and -minor axes of the ellipsoids. Mehl et al. (1940) have tabulated the numerical values of Y covering a wide range of p and q (Table VIII of the Appendix). It is noted that the viscosity increment is a function of the axial ratios only and does not depend upon the absolute molecular size of the particles. Each Y vtllue however corresponds to two axial ratios, p and q. A choice between the prolate and oblate models cannot be made from the viscosity measurements alone. For a homologous series of prolate ellipsoids having the same minor axis Hq. ( l l a ) can be approximated as (Simha, 1945) Y =
0.233 pl'"*,
20
5p 5
100
or
(llc) Y =
0.207 p'
13',
20
5
p I 300
Thus, Y should increase with the molecular weight or length to the 1.7 power. For cylindrical rods, the suggestion has also been made to substitute 1.8 and 0.8 for 1.5 and 0.5 in the denominators of the two terms in Eq. ( I la) (Sadron, 1953a), presumably on the basis of Burgers' theory ( 1938), for cylindrical rods. For very large p values such modification is insignificant, although Burgers' treatment has now been found inadequate (Haltner and Zimm, 1959; Broersma, 1960). Independently Kuhn and Kuhn (1945) obtained the following viscosity
333
VISCOSITY OF MACROMOLECULES
equations for rigid rods and discs: v = 0.4075(p
- 1)'
v = p2/15(10g, 2p
508
+ 2.5,
- 1.5)
+ p2/5(10ge2p - 0.5)
1
< 15 + 1.6, p > 15
(12a) (12b)
and v = ( 3 2 / 1 5 ~ )( ~1)
- 0.628(~- l ) / (-~ 0.075)
+ 2.5,
Q
>1
(12~)
It is noted that Eqs. ( l l a ) and (12b) only differ slightly in the constant term which is negligible for large axial ratios. By using as a model a linear array of spheres for the rodlike particles Kirkwood and Auer ( 19511 have calculated the intrinsic viscosity of these rods as a function of the axial ratio. (The relationship between intrinsic viscosity and viscosity increment is to be mentioned in the next section.) The asymptotic form of their equation is: [q] = ~NL'b/2250Mo loge ( L / b )
(13)
where N is t,he Avogadro number, L the length and 0 the radius of the rodlike particle, and M o the monomer molecular weight. It can be shown that for large axial ratios Eq. (13) is essentially identical with Eqs. ( l l a ) and (12b). Simha's equation [Eq. ( l l a ) ] , however, has been used most extensively although it is only recently that its experimental confirmation seems to have been definitely achieved.
B. Determination of Particle Shape from Intrinsic Viscosity Since the volume fraction, @, of the solute in Eq. (9b) cannot in general be determined experimentally, it has been a common practice to express the viscosity of Eq. (11) in terms of the intrinsic viscosity by the relationship [q] =
lim ( q -
c-0
qO)/voC' =
V,,v/lOO
(14)
noting that is equal to V,,C/lOO. Here Vspis the specific volume of the solute in solution, the over-all volume occupied by 1 gm of the (dry) solute plus that due to bound solvent. The concentration C in grams dry solute per 100 ml solution can be calculated from straightforward analytical procedure. However, since VBPis not measurable experimentally, the solution of Eq. (14) with two unknowns, V,, and v, thus becomes indeterminate. To circumvent this difficulty it is customary to replace Vapby the experimentally measurable partial specific volume, of the solute. Equation (14) then becomes
v,
[v]
=
vv/lOO
(with no solvation)
(15a)
334
JEN TSI YANG
This still does not solve the problem if the molecules are known to be hydrated (solvated) mainly because the volume occupied by the hydrated (solvated) particle will obviously differ from that of the dry particle. Mathematically, consider the volume of the solution as a function of the concentrations of dry solute, m, and solvent, mfl, the latter being the sum of the free solvent, m’o, and bound solvent, my . Thus I/‘ = V(m, mo) = ~ ( mmi ,
+ my)
Let dmb’ = wdm where w is the grams of solvent bound to 1 gm of solute. We then have by partial differentiation
dV
=
[(aV/am),,
+ w(aV/amo),]dm + (aV/amo),dmk
and at constant free solvent,
(aV/am),;
=
(aV/arn),,
+w(av/ad,
or
( a v / a m ) , ;=
vo
P + wVfl = 7 + w / p
(16)
where is the partial specific volume of the solvent which can be considered as the reciprocal of its density, p. The term on the left side of Eq. (16) turns out to be identical with the partial specific volume of the hydrated (solvated) protein on the basis of dry protein mass, P h . The reason is as follows: imagine that one can first prepare a hydrated protein and then dissolve it in a large quantity of free solvent. Under these conditions no additional free solvent will be bound to the already hydrated (solvated) protein molecules, i.e., the free solvent, m i , remains constant. Thus the over-all increase in volume of the solution per unit (dry) protein mass represents PJ,. Accordingly Eq. (16) can be rewritten as 8 h
=
P
+ w/p
=
iql
+ w/Bp)
(16a)
Following Oncley (1941) one can rewrite Eq. (14) as [q] =
7h;av~,/lOO= 8 ( 1
+ w/vp)vh/lOO
(with solvation)
(15b)
In the above derivation the bound solvent is assumed to have the same composition as the free solvent. This may not be exactly true for proteins in salt solutions, although the amount of salt involved in the bound solvent is so small that the resultant errors are quite insignificant (Tanford and Buzzell, 1956). Despite popular misunderstanding, Eq. (16) is derived without first assuming that V,, represents the sum of two volumes, one corresponding to the dry protein and the other the bound solvent. To substitute for V,, in Eq. (15a) obviously P must be positive and a nega-
VISCOSITY OF MACROMOLECULES
335
tive value has never been reported for proteins. On the other hand, to use Eq. (15b) for hydrated proteins it is immaterial whether P in Eq. ( l e a ) is positive or negative as long as the sum of 7 and w/p is positive. It is generally recognized that proteins are hydrated in aqueous solutions, but the precise ranges of hydration are still little understood and not well characterized. To quote Edsall (1954), the lower and upper limits of the degree of hydration probably lie between 0.04-0.1 and 0.6-1.1 gm of water per gram of protein. In the hydrodynamic sense this water of hydration should not only include that tightly bound to the molecules but also that loosely dragged along with them. In any event the estimate of the axial ratio from Eq. (15b) is made possible only if the quantity 20 is known in advance. Oncley (1941) employed the assumption that:
V(P)
= (1
+ W/PP)VA(P)
(15c)
and designed a contour diagram in which the axial ratio, p, is plotted against the degree of hydration, w, with v as a parameter. Thus with known [ q ] , P, and thereby v [Eq. (Ma)], one can calculate the V h values for various w values and the contour line of this particular v describes the range of possible p values. The usefulness of this treatment lies in the fact that the 7is probably very close to the specific volume of the dry protein and w is assumed to vary over a not too large range. Therefore Eq. (15b) provides a rough estimate of the shape of the proteins. Let us now examine the assumptions underlying the Oncley treatment’. First, thermodynamically P is defined as the increment of the volume of the solution per unit mass of the solute added and therefore is not identical with V,, of the solute. These two quantities may be equal in magnitude if and only if the system is an ideal solution, that is, there is no solute-solvent interaction whatsoever present. To eliminate one unknown V,, in Eq. (14) by introducing we have at the same time added another uncertain term w into the equation. Thus this treatment offers at most a rough estimate of the shape of proteins for a chosen model, a prolate or an oblate ellipsoid. Furthermore the estimated p value corresponds only to the hydrated particle, which is slightly different from that of the unhydrated particle unless the bound water is so distributed throughout the protein molecule that it does not change its axial ratio because of hydration. Secondly, the use of Simha’s viscosity increments for protein molecules implies automatically that the shape of the molecule can be approximated by an equivalent hydrodynamic ellipsoid having the same volume as that of the protein. The uncertainty involved in this second assumption is not just a problem of hydration. Even if the degree of hydration were precisely known and even if 7and Vsp were identical the axial ratio as determined from the intrinsic viscosity may still not represent the real molecule.
336
JEN TSI YANQ
Mathematical treatments such as Simha’s are exact, but the simple fact is that the actual shape of a protein molecule could be far from any fictitious model. The particle might be very irregular in shape and it might not even be perfectly rigid. The mathematical theories simply cannot handle completely the real physical situation, and the models are so chosen that they can deal with it in not too complicated a fashion. Thus all one can hope for is to deduce from the hydrodynamic measurements the dimensions of an equivalent model which behaves hydrodynamically as if i t were the actual molecule. It seems rather unfortunate to find that some workers have a tendency to accept Eq. (15b) without reservations as if it can give axial ratios even accurate to the first decimal. The truth is just the opposite. If there is good agreement between the hydrodynamic properties and other physical measurements, it may imply that the chosen model is probably not too far from the actual picture. On the other hand inconsistent results may actually shed some light on the real shape of the molecule as distinguished from a simple mathematical model.
C. Other Hydrodynamic Properties For the sake of discussions in a later section we will summarize here the theoretical equations of two other important hydrodynamic properties, the translational and rotary frictional coefficients. The former, designated as J can be determined from either sedimentation (Svedberg and Pedersen, 1940) or diffusion (Einstein, 1905, 190Ab; Smoluchowski, 1906) measuremrnts: fa
=
M ( l - Pp)/Ns
( 1 7%)
fd
=
kT/D
(17b)
and HereM is the molecular weight and P the partial specific volume of the solute, N the Avogadro number, k the Boltzmann constant, and T the absolute temperature; s and D are the sedimentation and translational diffusion coefficients (after extrapolation to infinite dilution). The translational frictional coefficients from both measurements are regarded as identical, i.e., fa = fd . The rotary frictional coefficient, designated as {, can be determined from either flow birefringence or non-Newtonian viscosity measurements. For ellipsoids of revolution Perrin (1936) has shown that the translational frictional coefficient is given by the equation:
Do/D
f/fo
=
(1 - q2)1’2/42’3 log,([l
for a prolate model ( q = b / a
+ ( 1 - 42)1’2]/9)
(18%)
< 1), and
Do/D = f/fo = (q2 - l)”2/q2’3arctan (in radians) (q2 - 1)”’
(18b)
337
VISCOSITY OF MACROMOLECULES
for an oblate model ( q = b/a > 1). [Very similar equations have also been derived by Herzog et al. (1934).] Here f/fo is known as the frictional ratio and its numerical values as a function of the axial ratio are given by Svedberg and Pedersen (1940), Cohn and Edsall (1943), and Sadron (1953b). (Table I X of the Appendix.) Symbols fo and Dorefer to the frictional and diffusion coefficients of a sphere of the same volume as the ellipsoid and can be calculated from Stokes’ law:
kT/Do
= fo =
6 ~ 7 0=~6 ~ 7 0 ( 3 V / 4 ~ ) ” ~
(18c) where r is the radius of the equivalent sphere, and V the volume of each particle. Thus the frictional coefficient,f, depends on both the volume, V , and axial ratio, ( p = l/q), of the particles. According to Perrin (1934), the rotary frictional coefficient, {, and thereby the rotary diffusion coefficient, 8, for ellipsoids of revolution can be given as
for a prolate model, and ~
0= ,
=
(3/2)([qz(q2- 2 ) / ( q z - 1)1/2~ arctan (in radians) ($’ - 1)‘”
+ $}/ti
-
1)
(19b)
for an oblate model. [Gans (1928) has also derived a set of formulas which are not exactly identical with Perrin’s, but such that the numerical values of 0 calculated from both equations are nearly the same.] The numerical values of {/{a for various axial ratios have been listed by Scheraga and Mandelkern (1953). (Table I X of the Appendix.) Here {,, and 0, again refer to the coefficientsof a sphere of the same volume as the ellipsoid and can be calculated from Stokes’ law: kT/&
= {O
= 8?rq0r3 =
6qoV
Thus, like f the rotary diffusion coefficient, {, also depends volume and the axial ratio of the particles. For large axial ratios Eq. (19) reduces approximately to
(19c) 011
both the
qoeb/T = (3k/16za3)[2 log, ( 2 ~ / b ) 11
(19d)
and @Ob/1’
= (3k/16nb3) [arctan @ / a )
+ (a/b)]= 3k/32b3
(19e)
Equations (19d and e) have been extensively used in flow birefringence measurements and, more recently, in non-Newtonian viscosity studies. (For an ellipsoid there are three translational and rotary frictional coefficients,
338
JEN TSI YANG
fi ,f2
,f3 and {I, C2 , , each characterizing the resistance to motion of the ellipsoid parallel to one of its principal axes. For an ellipsoid of revolution, two of the f’s and {’s are identical respectively. The experimentally determined diffusion coefficient and frictional coefficient are given by the relationship :
+ + D3)/3
D = (01 Dz or
D
= kT(l/ji
D
=
+ l/fi + l/f3)/3
(18d)
or
kT/j
I n the case of rotary diffusion and frictional coefficients, only the rotation of the major a-axis about the minor b-axis is in general experimentally measurable. Accordingly only the equations for 6,and { b will be considered here. ) Following Oncley (1941) the frictional ratio for hydrated particles can be separated into two factors
f/fo
=
(f/fh)
w - 0 )
(20%)
where the second term on the right side of the equation, designated as the hydration factor, is given by
j*/fo =
(Bh/P)”3 =
(1
+ w/Pp)1’3
(20b)
(2Oc) Again Oncley has constructed a contour diagram in which the axial ratio is plotted against the degree of hydration on a double logarithmic scale with j / j o as a parameter. By assuming a w value one can immediately read off the p value corresponding to the calculated f / j h . By the same reasoning one may also describe the rotary diffusion coefficient as
and {o =
8~70(3PM/4sN)= 67aPM/N
(2lc)
D. Concept of Equiualent Hydrodynamic Ellipsoid In view of the uncertainties raised in the Onclcy treatment a different
VISCOSITY OF MACROMOLECULES
339
approach to the problem was proposed by Sadron as early as in 1942 although it did not then receive wide attention, probably owing to wartime conditions. More recently this analysis has been discussed in detail by Scheraga and Mandelkern ( 1953). These authors suggest the use of a rigid equivalent ellipsoid of revolution characterized by its effective volume, V , , and axial ratio, p , in the interpretation of the hydrodynamic properties of protein solutions. The shape of this fictitious model may not necessarily resemble the dimensions of the real particles at all. They have also correctly pointed out the necessity of a combination of two hydrodynamic measurements for the determination of both Y e and p . The interpretation of any single hydrodynamic measurement could sometimes lead to erroneous conclusions. For example, the increase in intrinsic viscosity upon denaturation could arise from either increasing asymmetry or increase in V , or both, although in the past such a change was regarded frequently in terms of increasing asymmetry as a result of unfolding of the polypeptide chains. Scheraga and Mandelkern specifically reject the implication of Eq. (16) as representing the specific hydrodynamic volume, because it ('neglects possible flow of solvent through the domain by the hydrodynamic forces, selected adsorption from mixed solvent, electrostriction, and similar effects," Instead they rewrite Eqs. (14), (18c), and (19c) as [q]
=
NVev/lOOM
(22)
fo = 6 ~ q o ( 3 V e / 4 ~ ) ~ ' ~
(23)
To = 6qoVe
(24)
and By combining [q] [Eq. (22)] with either s (or 0)[Eqs. (17a, b, Ma, b, 23)] or 0 [Eqs. (19a, b, 24)] and eliminating V , they have evaluated two functions designated as 0 and 6.
and = Jv
Here y = N"3/(162007r2)1/3, F = fo/f, and J = ~oO/C.Both the P- and 6functions depend on the axial ratio only and their numerical values are listed in Table X of the Appendix. Once the axial ratio is determined the
340
JEN TSI YANG
effective volume, V , , can be calculated from one of the equations, Eqs. (22), (2X), and (24), which in turn gives the major and minor axes of the ellipsoid of revolution. This iiew analytical approach has since become a controversial subject (see, for example, Tanford and Buzzell, 1954, 1956; Loeb and Scheraga, 1956; Tanford, 1957a; Scheraga and Mandelkern, 1958; also Lauffer and Bendet, 1954). One argument is concerned with the use of an effective hydrodynamic volume. For years we have been accustomed to the Oncley treatment as a convenient method for estimating the axial ratios of the proteins. Now this new analysis raises serious doubts about the use of partial specific volumes and therefore seemingly uproots the very foundation of the conventional treatment. Naturally one may wonder what can be gained by characterizing this equivalent ellipsoid the shape of which might be quite different from the actual particle, and therefore the calculated values of V , and p might lack any physical significance. This argument actually points up the major contribution of this new concept which recognizes the uncertainties involved in t,he conventional oversimplified treatment and warns against its indiscriminate use that could lead to illusory conclusionx (see Section 111, B ) . (The word “effective” is introduced to emphasize the nature of this equivalent ellipsoid which effectively represents the hydrodynamic behavior of the real molecule. It would be redundant to call the exact mathematical models “effective,” but by the same token it is questionable to regard any actual molecule as corresponding to an exact model.) Another oft-repeated argument involves the expression of Eq. (16). Scheraga and Mandelkern reject it on the grounds that it incorrectly interprets (1) Q as the specific volume of the dry protein and (2) V, as the sum of two volunies, one for the dry protein and the other for the bound water. The second reason actually has nothing to do with the argument, since Eq. (16) can be derived without such an assumption. In this respect Lauffer and Bendet (1954) have argued for the use of v h instead of V s p (for hydrated proteins) by considering the viscosity as a function of the total amount of liquid displaced by the particles. But the real problem is and V,, have the same numerical values which could be not whether almost true a t least for the protein solutions. Rather, the question is: can we equate the particle volume with its hydrodynamic volume? It is the acceptance of this equivalence without reservations that has been disputed convincingly by Scheraga and Mandelkern. It is somewhat unfortunate that the current controversy has overshadowed the real test of the new analysis which at present is still lacking. Both the conventional and new analyses assume an equivalent ellipsoid and attempt t o solve for two unknowns, the volume and the axial ratio. Actually Oncley has also pro-
vh
VISCOSITY O F MACROMOLECULES
34 1
posed to combine tJwo contour graphs from viscosity and sedimentation (or diffusion) and determine t,he overlapping portions of the two sets of curves (after taking experimental errors into consideration) which define the possible range of the axial ratios (see, for example, Mehl et al., 1940). It caii be shown that by so doing the conventional treatment has implicitly eliminated the volume term, v(1 w / v p ) , just in the same manner as the p-function which, however, removes the volume restriction as defined in Eq. (15b). The shape of these curves is such that they never cross sharply, a fact merely reflecting the insensitiveness of the axial ratio determinat.ion from this procedure. The same difficulties are encountered in the
+
103
10'
10
bla AXIAL
t
I
10 10' n/b -+ RATIO
10'
FIG.1. Viscosity increments, Y, translational frictional coefficients, f/fO , arid @-functionsof the ellipsoids of revolution at various axial ratios. Left: oblate; right: prolate.
use of P- arid &functions. The reason is simple. Any hydrodynamic eyuatioii contains a product of (volume X shape factor) and by eliminating the volume from a combination of two hydrodynamic measurements only the ratios of the t>woshape functions remain in the final equations. The insensitiveness of the latter is therefore not unexpected even though each shape function varies significantly with the axial ratio. This can best be illustrated graphically in Figs. 1 and 2, where the viscosity increment, Y, and the frictional coefficient,s,f/joand { / T o , are plotted as a function of the axial ratio, p , and compared with the corresponding p- and &functions. Thus the intrinsic viscosity increases and the sedimentation coefficient decreases (i.e., the translational diffusion coefficient increases) with increasing axial ratio, but the product ~ [ 7 7 ] ~ in ' ~ the @-function does not increase much over the same range of axial ratios. For example, if the experi-
342
JEN T S I YANG
mental p is 2.15 f 0.06 X lo6 it is not possible to ascertain whether the particle is a sphere, or an oblate ellipsoid having an axial ratio of almost any value, or a prolate ellipsoid having an axial ratio less than five. The situation is somewhat better for the &function, but unfortunately the experimental rotary diffusion coefficient is also more uncertain than the sedimentation coefficient. Thus both functions can only give a rough estimate of the axial ratio even if the experimental data are of the highest possible precision. With axial ratio determination uncertain it becomes meaningless to calculate the effective volume and the major and minor axes. The p-func-
t
t
I
%. AXIAL
,
,
, a/b
,
-.
RATIO.-
r/r~
FIG.2. Viscosity increments, Y, rotary frictional coefficients, , and b-functions of the ellipsoids of revolution at various axial ratios. Left: oblate; right: prolate. Notice the difference in scales between Figs. 1 and 2.
tion however has found an unexpected use in molecular weight determinations. With known 1771 and s and by assuming a reasonable p value the molecular weight as calculated from Eq. (25) usually agrees very well with that obtained from other methods. One fundamental question concerning the new approach seems to have passed unnoticed despite the many arguments that have been raised aginst it. In the derivation of the p- and b-functions, Scheraga and Mandelkern have assumed that the three hydrodynamic properties can be represented by an identical equivalent ellipsoid. If either V , or p of this fictitious ellipsoid is arbitrarily fixed the other is automatically defined. But there is no a priori reason tjo assume t,hat the particles under shearing stress and sedimentation should fit the same hydrodynamic model. If V , is kept identical the appropriate p value for describing one property might differ from the
VISCOSITY OF MACROMOLECULES
343
appropriate value for the other and vice versa. If the particles are very rigid and resemble some simple model, say, a cylindrical rod, one might expect a certain simple relationship between the actual particle and its equivalent ellipsoid. On the other hand if the particles have very irregular shapes and if they are not perfectly rigid and solvent-impenetrable one cannot accept the equivalence of the two ellipsoids under shearing stress and sedimentation without serious reservations. This is based on the fact that the motions of the particles could be quite different in the two measurements. Suppose, for example, that the particles have holes which let the solvent molecules penetrate freely. Conceivably there will be a constant backflow of the solvent through the interior of the particle under sedimentation, whereas the particles which rotate under the influence of velocity gradient will merely drag the solvent along. The same argument has been well discussed by Flory in his treatment of flexible coils (see below). The differencebetween the two equivalent ellipsoids might be very small. Nevertheless it seems just as dogmatic to disregard this point as to accept the arbitrary restriction of P = V,, in the conventional treatment. I n particular since the ,&function is so insensitive to the variation of p any small deviation from equivalence of the two ellipsoids could manifest itself significantly in the final determination of the axial ratio and the effective volume. In this respect the &function should in principle be more reliable since both [q] and 8 can be determined under the same applied shearing stress. If the particles possess a certain degree of flexibility Scheraga and Mandelkern (1953) have also noted another complication owing to the possible deformation of the particles. Under these circumstances the equivalent ellipsoid as determined at any finite velocity gradient may not necessarily be the same as that at zero gradient. This discussion does not cast doubt on the concept of the equivalent ellipsoid. It merely emphasizes the necessity of extensive tests of the /3- and &functions. The new approach has sharpened our understanding of the hydrodynamic properties and compelled us to take a critical look at the interpretations of the experimental data, which in itself is a significant contribution. The foregoing discussion seems to give a rather pessimistic point of view. On the one hand the conventional treatment is now in serious doubt and on the other the new analysis still leaves much to be desired. This however does not mean that one can obtain no information at all about the shapes of proteins in solutions. It merely implies that one can never be too critical about the interpretations of the experimental results. Viscometry is, and will always be, a powerful tool in the studies of proteins and other macromolecular systems. It is most useful in the studies of conformational changes such as protein denaturation, degradation, and aggregation. It will continue to be valuable in providing us with a rough picture of the general
344
JEN TSI YANG
shape of the protein molecules. The Oncley treatment has played an important role in the past in spite of its many shortcomings. It seems certain that this procedure will continue to be used by many workers because of its simplicity. But it will be most unfortunate if one takes its conclusions too literally and ignores completely the arguments that have been raised against it. This is particularly true in the studies of protein denaturation where the application of the Scheraga-Mandelkern analysis will often allow us to deduce whether the change in intrinsic viscosity upon denaturation is primarily due to a change of the shape or the effective volume or both, even though it may be only of semiquantitative nature. However, this new approach, sound in concept, has yet to be subjected to extensive tests. Until this is done it seems rather drastic to condemn the use of the conventional treatment as completely out of date, especially since the 0- and &functions do riot guarantee any better and more precise determination of the shape of proteins. There is a tendency to put too much emphasis on the differences rather than the similarities of the two treatments, thus causing a rather nnneressary controversy. 13. Estimation of the Length of Equivalent Ellipsoid
Although both the p- and &functions are too insensitive and uncertain for the determination of axial ratios, the length, L, of the equivalent ellipsoid (prolate) can be calculated with confidence from either the intrinsic viscosity a t zero gradient or the more commonly known flow birefringence. This can be shown as follows: Eq. (22), (19a and c ), arid (18a and c) can be rewritten as
[VIM = (4rN/300) (a3)bh’)
(27a)
V O ~= / T(k/’%)( 1 b 3 )( J p ’ )
(28%)
and
f
=
( A ~( a~)( 1~ / ) ~ ~ ~ / ~ )
(2%)
which in turn become
L = 2a
=
I: = 2a
= 3.53
L = 2a
=
G.82 X 10-S([~]M)1’a(p2/y)1’3
x
(27b)
1 0 - 6 ( ~ ~ ? t a 0 ) 1 1 3 ( ~ ~ p 2 ) 1 ’ 3 ( 2%
1
and 1.76 X 10-26[M(1- ~ p ) / 9 0 s ] ( P p ~ ~ ~ )(20b)
( L is expressed in centimeters.) In Table XI of the Appendix are listed the values of ( p ’ / ~ ) ~ ’(Jp’)113, ~, and (8‘~’’~)which for large axial ratios, p , can be approximated as
345
VISCOSITY OF MACHOMOLECULES
( p 2 / v ) ” 3 = constant
x
( JP’)’’~ = ( 3 log, 2p
p’”’,
- 1.5)1’3,
> 20
(27c)
p
>5
(28c)
p
>5
(29c)
p
and
Fp2’3 = log, 2p,
Thus at least for intrinsic viscosity at zero gradient and flow birefringence appreciable errors in the calculated lengths are unlikely to result if a somewhat incorrect p value is assumed in Eqs. (27b) and (28b). In fact it has been shown that the viscosity method (at zero gradient) [(Eq. 27b)I is as good as the more common flow birefringence technique and both methods given entirely comparable errors for the IIRC: of incorrect p values (Yang. 1961b). Flow birefringence is known to be extremely sensitive to the degree of polydispersity of the rigid particles as reflected in the variations of the calculated lengths with velocity gradient. Precisely because of this it is very difficult to define a mean length for a polydisperse system. This difficulty is not encountered in the viscosity method (at zero gradient) and the viscosity-average length is usually regarded as close to the weight-average value. Unlike flow birefringence the viscosity measurements are simple, precise, and usually less time consuming. Another difference is that flow birefringence becomes impractical if the partihes are not very elongated whereas the viscosity method (at zero gradient) as discussed here is subjected to no such limitation so long as the axial ratio of the particles is fairly large. The requirement of a known molecular weight in Eq. (27b) seems to be a disadvantage of this viscosity method, although this quantity is usually available in the characterization of any macromolecule. As an illustration the calculated lengths of several proteins and polypeptides using Eq. (27b) are listed in Table 111. The agreement between viscosity and other methods is self explanatory. According to Eqs. (27b), (28b), and (29b) and Table XI of the Appendix, the use of an assumed axial ratio larger than the true value always results in a positive deviation in the estimated length. This in turn will reduce both the minor axis (or diameter), 2b, and the effective volume, V , . An upper limit of the p value can however be estimated from the Oncley treatment, since it is very unlikely that V ecan be much less than M v / N . Similar calculations can be given for oblate ellipsoids, but the errors will be larger than those for the prolate ellipsoids if an incorrect p value is used. In any case the flattened disc model rarely finds use in biological colloids. The same uncertainty has also been found in the use of Eq. (29b) and it will therefore not be discussed further.
JEN T61 YANG
346
F . Theories for Flexible Coils Theoretical treatments on the viscosity of solutions of polymer chains are too numerous to give even a brief summary. Originally their principal objective was to explain the intrinsic viscosity-molecular weight relationship as described in Eq. ( 5 ) . Now the major interest goes far beyond that and toward a better understanding of the solution properties of polymers. Our brief discussion will be confined only to general terms. The approach TABLEI11 The Calculated Lengths of Several Proteins and Polypeptides from Intrinsic Viscosity at Zero Shearing Stress Proteins and polypeptides Fibrinogen Tobacco mosaic virusb Myosin Collagen Poly-7-benzyl-L-glutamates M , = 130,000 M, = 208,000
From light From flow birefringence scattering (A) (A)
Range of axial ratio assumed
From [do (A)
10-20 15-25 30-70 100-200
580420 3450-3710 1600-1740 2760-2890
62O-68Oa 3340-3530'
50 80
920 1510
-
-
2400-3000'
-
3000" 1620d 3000' 910, 1410f
Scheraga and Laskowski 4957). The calculated lengths for this virus, as obtained from [ q ] and ~ from flow birefringence, require revision to take account of the fact that the virus is more accurately represented by a rod of uniform thickness than by an ellipsoid of revolution. When due allowance is made for this, the calculated lengths are very close to those determined by electron microscopy. See Haltner and Zimm (1959) and the discussion in Section 111, H , 4 below. 0 Boedtker and Simmons (1958). Holtzer and Lowey (1956,1959). Boedtker and Doty (1956). f Doty et al. (1956). a
adopted by Brinkman (1947) and Debye and Bueche (1948) is to replace the actual polymer coils by a porous sphere having an effective radius, R. The effective porosity of this equivalent sphere is expressed by a shielding ratio, RIL, where L represents the depth to which the solvent can almost freely penetrate into the sphere. This model is so general that it does not depend on the particular assumed molecular structure, such as Gaussian or non-Gaussian chains, branched chains, etc. A more realistic and rigorous treatment was first developed by Kirkwood and Riseman (1948) using a necklace model and taking into consideration all the intramolecular interactions. I n both approaches tlhe solvent is regarded as a n "inert" continuous medium and its only influence on the intrinsic viscosity manifests itself
347
VISCOSITY OF MACROMOLECULES
through certain parameters such as the frictional coefficient which in turn is related to the permeability of the solvent. An alternative approach has been formulated by Flory and Fox (1951) (see also Flory, 1953) who take into consideration the excluded volume effect and introduce an “equivalent hydrodynamic sphere,” the radius of which is postulated to be proportional to the root-mean-square end-to-end distance, (r2)l/’,or the radius of gyration, (s2”/’ [Note that (r’) = 6(s2) (for a Gaussian chain).] I n all these treatments the intrinsic viscosity can be expressed as [TI n: (T’)~/’/M= ( S ~ ” ~ ’ / M
(30)
In Flory’s treatment the term (r2)3’2/Mis rearranged into ( ( T : ) / M ) ~ ‘ ’ M ~ ’ ~ ~ ~ , where (r:) refers to the ideal, unperturbed state. The expansion factor a (not to be confused with the same symbol used in Section 111, A ) is defined as ( T ~ ) ~ ’ ~ / ( T which ; ) ~ / ~ reduces to unity in an ideal solvent or, in Flory’s terminology, at the &temperature.’ Equation (30) is very similar to that for rigid particles [Eq. (22)], since both (T’”’~ and (s’”’’ are proportional to the hydrodynamic volume of the polymer chain. Unlike the equations for rigid, impermeable particles those for the viscosity of flexible polymers do not contain a shape factor such as Y, but the hydrodynamic volume is dependent on the solute-solvent interaction, In the limiting case of complete solvent immobilization inside the molecule (according to Debye and Kirkwood) or at 0-temperature (according to Flory ) the above-mentioned treatments give essentially identical results (see also Peterlin, 1959). Debye-Bueche:
[T] =
22
2 312
13.57 X 10 ( s ) / M
Kirkwood-Riseman-Zimm:
4.17 X 1022(~2)3/2/M(30a)
Flory :
3.7
x
1022(~2)312/~
2 The osmotic pressure, H , of dilute polymer solutions may be expressed as a function of the concentration, C , in the form
T/C = RT(B1
+ BzC +
. a * )
(R = gas constant and T = absolute temperature). The so-called first virial coefficient, BI , is simply equal to the reciprocal of the molecular weight, M. The evaluation of the second virial coefficient, B Z, has become one of the principal theoretical developments in recent years. In his theory of polymer solutions Flory (1953) has shown that B I is proportional to a term (1 - e/T), which vanishes a t T = 8. Accordingly e may be considered as the “ideal” temperature a t which the above equation is reduced to the well-known van’t Hoff’s law, i.e., H / C = RT/M To avoid possible confusion with the symbol for rotary diffusion coefficient here we have used the symbol e for the theta temperature, rather than 0 which was originally used by Flory.
348
*
JEN TSI YANG
The numerical constant, of the Kirkwood-Riseman equation was originally and later reduced to 4.8 X 10” ( Kirkwood et al., 1955). given as 5.3 X A further revision by Auer and Gardner (1955) gives a valw of 1.25 X loz2 which agrees well with Zinim’s 4.17 X 10’” (1956). The experimental value by I’lory and his co-workers has risen from the original average 3.1 X 10“ to a more recent value of about 3.7 X loz2.Krigbaum and Carpenter (1955) reported that the Flory constant actually increases with decrease in the second virial coefficient (see footnote 2). A theoretical explanation for this variation for systems close to the 0-temperature has been given by Kurats and Ysmakawa (1958) and Yamakawa and Kurata (1958). The apparently too-large-value of the Debye-Bueche theory probably reflects its unrealistic assumption of a uniform segment distribution inside the porous sphere which underestimates the hydrodynamic interac tion in the coils. Nevertheless it is indeed surprising to find the closeness of various values despite the use of different models. Altogether both theories and experiments seem to converge to a good agreement at least in this limiting (we. Similarly, the theoretical treatments of the sedirne~it,ationfor. romplete solvent immobilization or at 0-temperature ran be written as: Debye-Bueche :
s
=
(1/24.3) M ( 1 - P ~ ) / N T o ( s ~ ) ” ~
Kirkwood-Riseman :
(1/12.73) M ( l - P ~ ) / N ~ O ( & *(81) ’*
Flory :
(1/12.56) M ( 1 - B ~ ) / N ~ I o ( s ~ ) ” ~
By combining Eq. (30a) with Eq. (31) and eliminating the term s2 we have : Debye-Bueche : Kirkwood-Riseman : 1i‘ior.y:
~~[~]*/~~~ /Pp) n i ~= /2.12 ~(x 1 10‘ 2.72 2.10 (theor.)
(32)
2.5-2.7 (expt,l.)
It is not,ed that the left side of Eq. (32) is exactly identical in form with the &function for rigid particles (Section 111, 0). I n lglory’s treatment the numerical coefficient on the right side of the equation should be a universal constant, designated by him as $1’3P-1. This constancy has been confirmed experimentally for many polymers studied, although the experimental values are always higher than the theoretical one. This has been attributed by Flory to the possible difference in the equivalent spheres under shearing stress and under sedimentation. It is also interesting to note that this constancy applies to those highly extended polymer rhsiris such as celliilose
VISCOSITY OF MACROMOLECULES
349
derivatives even though the coefficients for viscosity and sedimentation in Eqs. (30a) and (31) are no longer constant separately. With increasing solute-solvent interaction or a t temperatures other than the &temperature Flory’s theory still predicts a constant coefficient in Eq. ( 3 2 ) ’ whereas according to the Debye-Bueche treatment the increase in solvent permeability results in an increase in the coefficient in the same manner as the ,&function increases with the axial ratio. This raises a n interesting quest,ion concerning the use of the p-function for rigid particles (Section 111, D ) . A constant value of 2.5 X lo6 according to Flory will correspond to an axial ratio of 15 from the p-function for a prolate ellipsoid, not to mention the unavoidable experimental errors. Since the theories for linear polymer chains are not directly applicable to most proteins it is not possible a t present to predict whether the coiled form of, say, a denatured protein having many cross-linkages would give a p-value closer to 2.1 X lo6 for a hard sphere or 2.5 X 10’ for flexible coils or even to some other values. This problem can only be clarified by extensive experimental analyses. Thus in the absence of any information concerning the rigidity of the molecules under study there is reason for caution in the quantitative interpretation of the &function. Branching. So far our discussion has been limited to linear polymer chains. The effect of branching on viscosity is still not well understood. The statistics of certain simple types of branched chains has been studied by Zimm and Stockmayer (1949) and Stockmayer and Fixman (1953). Since branching produces a less extended hydrodynamic volume than would be expected for a linear chain of the same molecular weight, conceivably the intrinsic viscosity for a branched polymer would be smaller and the sedimentation coefficient larger than those for a linear polymer. At present quantitative treatments are still scarce.
G. Polyelectrolytes and Electroviscous E$ect By definition polyelectrolytes are a class of macromolecules having ionizable groups distributed at intervals along the polymer chains. I n the absence of any other restriction such as cross-linkages, a flexible coil possessing ionic charges of like sign expands into a more extended chain conformation as a result of electrostatic repulsion. This polyelectrolyte effect is therefore dependent, on the degree of ionization. It can be partially neutralieed by the distribution of ions of opposite charge (counter ions) and thus repressed by an increase in ionic strength. If ionic charges of both signs are present along the chain in approximately equal number the molecule tends to coil more tightly than if it were uncharged, mainly because of the strong electrostatic attraction among the oppositely charged groups. A polyelectrolyte such as denatured protein a t its isoelectric point is an example of this class.
350
JEN TSI YANG
The polyelectrolyte effect manifests itself in many of the solution properties of the macromolecule (for a brief review, see Doty and Ehrlich, 1952). The titration behavior of polyelectrolytes has been studied extensively, both theoretically and experimentally, and its discussion is beyond the scope of this review. Hydrodynamically, this effect also influences the measurements of sedimentation, diffusion, flow birefringence, and viscosity of the polymers. At sufficiently high ionic strength where the ion atmosphere is clustered closely around the macromolecule, so that the charges on the molecule are well shielded, the polymer behaves as if it were uncharged. The sedimentation coefficient however begins to fall with decreasing ionic strength as a result of the dragging along of the counter ions and also the expansion of the polyelectrolyte. The diffusion coefficient however will increase with decreasing ionic strength owing to the fact that the counter ions diffusing ahead of the polyelectrolytes induce an accelerating effect which more than compensates for the increase in the translational frictional coefficient of the expanded polyelectrolyte. For flow birefringence both the extinction angle and the birefringence increment are very sensitive to the molecular asymmetry of the polyelectrolyte. I ts “abnormal” behavior in salt-free solution is believed to be due to the same cause as that of the viscosity discussed below (Yang, 1958b). I n the absence of added electrolytes the reduced viscosity, rlSp/C,of a polyelectrolyte rises upon dilution in a striking manner as a result of the expansion of the polymer chains (Fuoss and Strauss, 1948; Hermaris and Overbeek, 1948; Kuhn et al., 1948). Empirically, Fuoss and Strauss have found that the viscosity data can be represented by the equations qsp/C = a
+ b/(l + c d C I
(33)
+B/di)
(34)
in the absence of salt and [tll = A(1
in the presence of salt, where a, b, c, A and B are constants. Here A is the intrinsic viscosity in the limit of infinite ionic strength, p. The term a is usually very small as compared with the last term in Eq. (33) and thus can be neglected. The numerator b is sometimes considered as the intrinsic viscosity of the polyelectrolyte in its most swollen state, although, strictly speaking, this is not true since the ss,/C - C plot passes through a maximum and curves downward rapidly as the concentration approaches zero. Most proteins do not behave like polyelect
351
VISCOSITY OF MACROMOLECULES
molecular weights, presumably because of the presence of cross-linkages, etc. in the proteins which greatly restricts their expanded volumes. Electroviscous Efect [for a recent review, see Conway and Dobry-Duclaux (1960)l. I n an ionic solution or suspension each charged particle is surrounded by an atmosphere of counter ions having a net charge of opposite sign to that of the particle. This ionic atmosphere undergoes deformation when subjected to an applied shearing stress, thus giving rise to a n additional dissipation of energy. This electrostatic contribution to the viscosity of the solution or suspension is commonly called the electroviscous effect. Smoluchowski (1916) was among the first to put forward a n equation, but without derivation, to explain this effect on charged colloidal particles. Twenty years later Krasny-Ergen (1936) derived an equation similar to Smoluchowski’s, except for a different numerical factor. Experimentally Bull (1940) studied this effect on the change in viscosity of ovalbumin with degree of ionization, but found that the experimental value was about ten orders of magnitude less than that predicted by the theory. This prompted Booth (1950, 1953) to derive a new equation for a spherical model which can be rearranged as (see, Tanford and Buzzell, 1956) [?]el
=
322[g][ 2z2$’ ( Ka )
( Cizz/xi)] / [ D kTgoU2
(C&)]
(35 )
where c = protonic charge, Z = charge on the particle, D = dielectric constant of the solvent, K = Debye constant (proportional to the square root of the ionic strength), a = radius of the sphere, $‘ = a decreasing function of KU (see Booth’s papers for its numerical values), ci = concentration of the ith component of the inorganic ions, Zi = charge of the corresponding ith component, and X i = ionic conductance in practical units. Comparison of Bull’s data with Booth’s theoretical prediction (under certain assumptions) appeared to be very favorable. Recently Tanford and Buzzell (1956) and Buzzell and Tanford (1956) further tested Eq. (35) with the viscosity data of bovine serum albumin and ribonuclease and found that the agreement between theory and experimental results was reasonably good. Some of Tanford’s calculations are listed in Table IV to illustrate the order of magnitude of this electroviscous effect. It is noted that the observed values were uniformly higher than the theoretical predictions, although the discrepancies were barely outside the experimental errors. On the other hand, these could be attributed partly to the nonspherical shape of the proteins which does not exactly fit with Booth’s simple model. Nevertheless the general opinion that the electroviscous effect is small for charged colloidal particles (Hermans and Overbeek, 1948) seems to have received strong support a t least in the case of globular proteins. Furthermore, this effect can be eliminated in the presence of high ionic strength. I n relatively high concentrations of polyelectrolytes where the double
352
JEN TSI YANG
layers and the charged particles overlap one another, a so-called second electroviscous effect may arise. These particles tend to move away from one another in the direction perpendicular to the flow. This mutual interaction (electrical repulsion) causes another extra dissipation of energy and therefore increases the viscosity of the solution or suspension. This contribution increases with the square and higher powers of the concentration a t constant ionic strength or with decrease in ionic strength at constant concentration. Frequently these particles also exhibit a non-Newtonian behavior mainly because the displacement of the encountering particles will be less the more rapidly t,hey pass each other. As a consequence the enhanced viscosity due TABLE1V The Electroviscous Effect Ionic strength
PH
5-5.6 5.0 7.3 7.3 5.9-11 2.8 2.8 9.6 b
0.014.05 0 0.01 0.05 0.01 0.05 0.15 0
-
Z
Bovine serum albumin0.037(mean) 0.0406 Ob -11 0.0412 -16 0.0383 Ribonucleasec 0.0330(mean) 0.0358 +16 0.0350 +16.5 O* 0.0336
Tanford and Buzzell (1956). At the isoianic paint is zero but Buzzell and Tanford (1956).
z
hl
(dl gm-l)
AM
Ah1
(obsd.)
(Booth Eq.)
0.004 0.004 0.001
0.0009 0.0008 0.0005
0.0028 0.0020 0.0006
O.OOO9 0.0004 0.0003
z*as used in the Booth equation is not zero.
to this electroviscous effect will be smaller at higher shearing stress. In general, however, this effect is not considered to be particularly strong with macromolecules (see, for example, Dobry, 1955).
H . Examples of Several Common Proteins and Polypeptides Viscosit,y data for proteins are indeed too numerous to give even a cursory summary. Rather we will consider only a few examples to illustrate the information that can be obtained from viscometry and the limitations in its interpretations in the light of the previous discussions. 1. Synthetic Polypeptides
Until recently the validity of Simha’s equation appeared not to have been subjected to unambiguous experimental confirmation, although it has been used extensively by the protein chemists. The recent investigations of
353
VISCOSITY O F MACROMOLECULES
synthetic polypeptides provide an ideal model for such a test. Doty et al. ( 1956) (see also Doty et at., 1954) found that poly-y-beneyl-L-glutamates exist in the form of Pauliiig and Corey’s a-helix (1950) (see also Yauling and co-workers, 1951) in solvents having low hydrogen-bonding properties.
.A P
-
L
0.05, l0,OOO
DCA BASED ON L.S. M, C-F BASED ON L.S. M, DMF BASED ON L.S. M, C-For DMF BASED on [TIocA M, ’
20,000
I
I
I I I100d,000
I
I I
200;000 Molecular weight
50,000
IIIll
600,000
1,oo
Fro. 3. Intrinsic viscosity-molecular weight relationship of poly-7-benzyl-Lglutamates. The open circles represent the randomly coiled form and other symbols the a-helical form. The line of steeper slope is plot of Simha’s equation. Abbreviations: dichloroacetic acid, DCA; chloroform saturated with formamide, C-F; dimethy1 formamide, DMF; light scattering, L.S.; weight-average molecular weight, M , . Reproduced from Doty ef al. (1956).
These authors have also shown that for a homologous series of polypeptides, the intrinsic viscosity varies with the molecular weight (from light scattering) to the power of 1.7,which is in perfect accord with Simha’s theoretical value (see Fig. 3 ) . By adopting the conventional treatment with P = 1/1.31 and w = 0 the [7]-M relation could be fitted to Eqs. ( l l a ) and (15a) for prolate ellipsoids
3 54
JEN TSI YANO
if the major axis of the hydrodynamic ellipsoid was equated with the length of the rod (with a pitch of 1.5 A per amino acid residue) and its minor axis was taken as 18.2 A (varying experimentally between 17.75 and 18.0 A in different solvents). Since the volumes of the rod and the equivalent hydrodynamic ellipsoid were assumed to be identical, the diameter of the former would become (2/3)''' of the minor axis of the ellipsoid. It turned out to be 14.9 f 0.3 A which agreed well with 15.0 A found from the X-ray study of Arndt and Riley (1955) for the average distance separating neighboring helices. Thus the conventional treatment seems to explain well the viscosities of the polypeptides. Such consistent results however did not prove the shape of the hydrodynamic ellipsoids and neither did they rule out other possibilities. It can easily be shown that if the axial ratios in the above calculations are changed by a constant factor the same [q]-M relation still holds, except that the hydrodynamic volume will differ from that calculated from V , = v M / N . Doty et al. (1956) also pointed out that the arbitrary equating of the rod and its hydrodynamic ellipsoid having the same length and volume can be avoided by using Eq. (13) for rods instead of Simha's equation. This however involves a similar arbitrary assumption in the calculations since the Kirkwood-Auer model is a rodlike array of spheres, thus requiring an adjusted radius to fit the experimental data. It, will also be interesting to find out what axial ratios the p-functions will yield for this simple model system, if the sedimentation coefficients were available. In Fig. 3 the intrinsic viscosity begins to deviate downward from the theoretical curve for rigid rods when measurements are extended to the molecular weight range of about one million. This was attributed by Doty et al. (1956) to the onset of a very small amount of flexibility for the long, thin a-helices. I n solvents having unusually strong hydrogen-bonding ability the intramolecular hydrogen bonds of the helices are no longer stable and the conformatlion of the polypeptides is that of a random coil. Doty et al. (1956) found that in dichloroacetic acid [77] = 2.78 X 10-6N0.87,the exponent falling in the range expected for flexible polymers. It should be pointed out that the curve for the coils in Fig. 3 could shift upward or downward depending on the solvents used. The two curves in the figure, however, cross and the intrinsic viscosity a t the intersection remains unchanged even if the helix is transformed into the coil or vice versa (see also Table I ) . On the right side of the intersection a shift from the helix to coil form is accompanied by a decrease in the intrinsic viscosity and the reverse is true on the left side of the figure. As a result of these studies Doty and Yang ( 1956) have also demonstrated a first-order helix-coil transition of polypeptides by measuring their viscosities or optical rotations in mixed solvents
355
VISCOSITY OF MACROMOLECULES
of ethylene dichloride and dichloroacetic acid. Similar reversible transition was also observed for poly-L-glutamic acid in 0.2 A4 NaC1-dioxane (2: 1) in the pH range of 5.4-6.4 (Doty et al., 1957). For moredetailsonthis subject seea comprehensive review in this volume by Drs. Urnes and Doty. 2. Serum Albumins
a. Native Conformation. Both human and bovine serum albumins have been the subjects of numerous physical and chemical studies. They are TABLE V Physical Properties of Serum A l b u m i n , Fibrinogen, and Tobacco Mosaic V i r u s Physical property Intrinsic viscosity, [Q] (dl gm-l) Partial specific volume, P(m1 gm-1) Sedimentation coefficient, ~ 2 ~ . , , , (Svedberg units) Translational diffusion coefficient, D z ~ ,(Fick , units) Rotary diffusion coefficient, €h rUI (sec-l) Molecular weight, M (gm mole-’) Molecular length, L(A) Light scattering Hydrodynamic a
Serum albumina 0.037-0.041 0.73
Fibrinogens Tobacco mosaic
virusc
4.4
0.25 0.71-0.72 7.7-7.9
0.37 0.73 188
5.9-6.1
2.0
-
40,000
300
330,000340,000
38,000,000-
-
500-600
-
500-600
3000-3400 3600
640,000d 65,50067,500
4 O O ,0 O m ,
Loeb and Scheraga (1956) and Champagne (1957).
a Quoted from Scheraga and Laskowski (1957).
Boedtker and Simmons (1958). Corrected from 036,w = 830,000 (O’Konski and Haltner, 1957).
known to be indistinguishable by most physical criteria. Some of the physical data are summarized in Table V. The shape of the albumins however is still a matter of controversy at the present time. These molecules were considered approximately as prolate ellipsoids ( p E 4) on the basis of the early work of Oncley et al. (1947). The choice of a prolate rather than a n oblate model was based largely on the conclusions drawn from the dielectric dispersion measurements (Oncley, 1942; Oncley et al., 1952; Dintzis, 1952), which gave two relaxat,ion times and could not, fit the data of any chosen oblate ellipsoid. Oncley’s analysis however ignored the contribution to the dielectric dispersion from polarization of the ion atmosphere about polyelectrolytes (see, for example, O’Konski, 1955; O’Konski and Haltner, 1957). There is another possible uncertainty in the dielectric measurement.
356
JEN TSI YANG
Kirkwood and Shumaker ( 1952) have recently given a new interpretation of the dielectric const,ants of protein solutions in terms of moments which arise from charge fluctuation due to proton migration among the various basic sites of the protein molecules. Such fluctuations can apparently contribute to the moments of the order observed experimentally for many proteins even in the absence of permanent, dipole moments. Whether this new theory offers a better account of the experimental results is stJillundecided. Nevertheless it casts some doubts on Oncley's conclusions. An entirely different conclusion seems to have come from the low angle X-ray scattering studies by Anderegg et al. (1955) and Luzzati (1960). At very low angles the scattering curves gave a radius of gyration which appeared to be much too great for a sphere. At somewhat higher angles the scattering curves seemed to be in better accord with an oblate model having an axial ratio of 3.5. X-ray data do not prove the structure and only suggest the compatibility of such a model with the experimental data. The discovery by Hughes (1947, 1950) of human mercaptalbumin mercury dimer has led Oncley to reconsider his earlier model in order to account for the steric relations for the dimer formation. This revised model consisted of a prism of approximately 144 X 45 X 22 A3, which fits well into the unit cell of the crystal as employed by Low (1952) in her X-ray diffraction study. As Edsall (1954) has pointed out t>heX-ray scattering function for the prismatic model might be quite different, from that of a prolate ellipsoid. Thus one still cannot rule out other mode!s which might agree equally well with the X-ray scattering result,s. Furthermore there also exists the possibility that a model which fits with one physical method may not be identical with the equivalent hydrodynamic ellipsoid. Loeb and Scheraga (1956) and Champagne ( 1957) have independently made careful studies of the hydrodynamic properties of bovine serum albumin, each from a single lot, which eliminated the possible complications that may arise from the use of different preparations. [The only value not, measured by these authors was the partial specific volume of the protein which was taken from Dayhoff et al. (1952) and corrected from 25°C to 20°C by using the general temperature coefficient for P for proteins (Svedberg and Pedersen, 1940).] Loeb and Scheraga have taken into consideration a small (3%) faster component in their sample whereas Champagne has found 8 % of dimer in her preparation. The physical data and the corresponding p-functions of the two groups are listed in Table VI for comparison. Independently Tanford and Buzzell (1956) found that [q] = 0.037 and, together with ~ 2 0 = , ~ 4.42 X from Creeth (1952), and M = 67,000, obtained a @-valueof 2.04 f 0.06 X lo6. Harrington et al. (1956) used [q] = 0.038, S ~ O =, ~4.45 X lo-", and M = 67,500 which gave a p-value
357
VISCOSITY OF MACROMOLECULES
of 2.05 x lo6. All four groups reached essentially identical results which are lower than the minimal theoretical value of 2.12 X lo6for rigid spheres. Loeb and Scheraga (1956) have claimed that the standard deviation cited by them was a minimum value which would be augmented by determinate errors in any of the measured values and they therefore do not regard this low @-valueas a failure of the theory. Champagne (1957) has also assumed a spherical shape for bovine serum albumin and attributed the lower than TABLEVI Calculations of the p-Functions of Bovine Serum Albumin Physical property PH Ionic strength Partial specific volume, P(m1 gm-1) Intrinsic viscosity, [ 7 ] (dl gm-1) Sedimentation coefficient, ~ 2 0 (Svedberg . ~ units) Translational diffusion coefficient, D z o , ~ (Fick units) Molecular weight, M (gm mole-') 8-Function X 10-6
Loeb and Scheragan
5.13 0.5 0.730b 0.041 (1) 4.41" (2) 4.48 (1) 5.90d (2) 5.81 (1) 67,000 (2) 69,200 (1) 2.05 f 0.06 (2) 2.04 f 0.06
Champagne"
5 0.15 0.730b (1) 0.0388 (2) 0.0607 (1) 4.42c (2) (1) 6.07 (2)3.89 (1) 66,400 (2) 161,000 (1) 2.04. (2) 2.04
a In Loeb and Scheraga's paper (1956) (1) refers to the major component and (2) the whole protein, whereas in Champagne's paper (1957) (2) refers to the minor component. Taken from Dayhoff el al. (1952). c Corrected for the adiabatic cooling effect (Waugh and Yphantis, 1952;Biancheria and Kegeles, 1954). d Taken from Wagner and Scheraga (1956). 0 Champagne has expressed her data in terms of a function v / F 3 (in our terminology), which differs from the p-function by a constant only.
theoretical value to experimental errors. But one also cannot rule out the possibility of an oblate ellipsoid as a model for the protein, since such an ellipsoid has a @-valuewhich is essentially independent of the axial ratio and is very close to that for a sphere. Following the same reasoning one may even suspect the possibility of a prolate ellipsoid of low axial ratio if the experimental errors were slightly higher than the standard deviation. Harrington el al. (1956) suspected that the anomaly was caused by approximations in the Simha equation for axial ratios less than 20. This however was refuted by Loeb and Scheraga (1956) on grounds that the viscosity increments, Y, in the p-function calculations are taken from the
:358
JEN TSI YANG
exact solutions of the Simha equation rather than from its approximate form. 1,auffer and Beiidet (1954) suspected that ambiguity might also arise from the lavk of precise knowledge of the theoretical equations such as Simha’s and l’errin’s, since any small deviations could lead to very erroneous p-values. On the other hand Tanford and Buzzell (1956) were of the opinion that serum albumin cannot be represented successfully by a solid equivalent ellipsoid, a suggestion which certainly should not be taken lightly. Indeed it would be less than critical to claim that the positive deviations of the p-values were more reliable than the negative ones in this case merely because the latter gave a lower than the minimum theoretical value and were therefore not acceptable. Since the serum albumins are known to have conformational adaptability and are perhaps not perfectly rigid even at the isoelectric point it is conceivable that the equivalent ellipsoid under shearing stress may differ from that under sedimentation. Such a discrepaiwy could result in an erroneous p-value. All one can say a t present is that the serum albumins are probably nonspherical, but that they :ire not, far from spherical. 6. Conformations in Urea Solutions and at Low p H . The denaturation of serum albumins by urea provides an example of the kind of information which one can draw from viscosity measurements and also the limitations of its interpretation. The increase in intrinsic viscosity and decrease in diffusioii roeffic.ient of horse serum albumin (Neurath and Saum, 1939) with increasiiig urea concentrations clearly indicated a marked change in the protein cmformation in such solutions. It had been commonly believed that protein denaturation involved the unfolding of the peptide chains, thus resulting in a molecule having a highly elongated rod shape. In terms of increasing asymmetry in urea solutions the axial ratio of the horse serum albumin was said to increase from about five in the native protein to about twenty in the denatured protein. Such interpretation based 011 any single hydrodynamic measurement is now known to be questionable, since it does not rule out the possibility of an increase in the effective volume rather than the change in shape. Scheraga and Mandelkern (1’353) found that the p-values of the above-mentioned results could be equally well interpreted in terms of a sphere and that the denaturation process involved an isotropic expansion of the molecule. True, the application of the p-function to nonrigid particles may still be open to question, but together with other information the inferences as drawn from the hydrodynamic meaburements seem to be very plausible in this case, although they may he only of seniiquantitative nature. Indeed Doty and Katz (1950) (see also Doty and Edsall, 1951; Kay and Edsall, 1956) concluded from their light scattering studies of bovine serum albumin in concentrated urea solutions that the principal change undergone by the albumin molecules is that
VISCOSITY OF MACROMOLECULES
3 59
of approximately isotropic swelling, although the details of their work are not yet published. The rapid reversibility of the urea denaturation of serum albumins as revealed from both optical rotation and viscosity measurements also manifest a lack of internal rigidity of the protein molecule (Kauzmann and Simpson, 1953; Frensdorff et al., 1953; Kauzmann, 1954). The concept of “configurational adaptibility” as proposed by Karush (1950) to account for the adsorptive power of serum albumins essentially leads to the same conclusion. Much attention has recently been focused on the behavior of serum albumins in acid media. Unlike most globular proteins serum albumins undergo marked structural changes at pH below 4. This is reflected in many physical measurements such as the abnormal sharpening of the titration curve (Tanford, 1952; Tanford et a,?., 1955a), increase in depolarization of fluorescence (Weber, 1952; Harrington et al., 1956), increase in intrinsic viscosities (Bjornholm et al., 1952; Yang and Foster, 1954; Tanford et al., 1955b), decrease in sedimentation coefficients (Harrington et al., 1956; Reichmann and Charlwood, 1954; Charlwood and Ens, 1957), and increase in levorotation (Yang and Foster, 1954). Bjornholm et al. suggested that the molecules aggregated end-to-end at low pH, thus implying an increasing asymmetry to account for the high viscosities, whereas Weber originally suspected a dissociation of the protein molecule into subunits to explain the depolarization behavior. Both interpretations seem now to be ruled out, since the light scattering studies (Doty and Steiner, 1949) indicate no change in molecular weight a t low pH. Both optical rotations and intrinsic viscosities of serum albumins were found to be extremely sensitive to pH and ionic strength in a manner analogous to the behavior of synthetic polyelectrolytes. Thus the overwhelming evidence seems to indicate an essentially isotropic swelling of the serum albumins in acid media. Here is just another illustration of the power of viscometry in detecting the conformational changes of proteins; but any single hydrodynamic measurement simply cannot give an unequivocal answer to the nature of such changes. (For more details on the subject of serum albumins see a recent>review by Foster, 1960.) 3. Fibrinogens
The human and bovine fibrinogens are also known to be remarkably siniilar in both size and shape. Their high intrinsic viscosity, strong birefringence, and significant angular dissymmetry of light scattering all indicate that the shape of the molecule is far from spherical. The interpretation of their physical properties has been complicated by the impure preparations employed in many iiivestigations except perhaps the most recqent ones. This subject has recently been reviewed by Scheraga and Laskowski (1957)
360
JEN TSI YANG
and the physical properties of fibrinogen in aqueous solutions are included in Table V. From electron microscope studies fibrinogen appears to resemble a string of beads and the lengths of the rodlike particles are in the range of 400 and 600 A (Hall, 1949, 1956; Siegel et aE.,1953). Mitchell’s (1952) preparation however seemed to consist primarily of spheres of 50 A in diameter and Porter and Hawn (1949) suggested a disk model for the protein. The diameter of the protein was found to be 3 0 4 0 A by Hall and 60-80 A by Siegel et al. The latter results would give a molecular volume two or three times that calculated from the specific volume of the protein. The electron microscope observations revealed considerable polydispersity for fibrinogen in the dried state, in direct contrast with the studies of solution properties which indicated that fibrinogen is fairly homogeneous. It is not yet clear whether this discrepancy could be attributed to artifacts which arose during the process of drying. The evaluation of the 8-function for fibrinogen in solution led to a very puzzling conclusion, i.e., that the axial ratio was only about five ! Edsall (1954) has considered the possible experimental errors and found that the lower and upper limits of 8 would be 2.05 X lo6and 2.28 X lo6.The former was below the minimum theoretical value for a sphere, whereas the latter corresponded to an axial ratio of six. This is in direct contrast t,o the weight of evidence available a t the present time and the explanation must lie elsewhere other than in experimental errors. As Edsall pointed out such an equivalent ellipsoid would indicate that the molecule is highly swollen by the embedded solvent and the effective volume would have been about five times that of the unhydrated molecule (assuming = V,,). A better explanation for such a large discrepancy between hydrodynamic and other physical properties is still lacking. Could it be that fibrinogen is represented by two different equivalent ellipsoids under shearing stress and under sedimentation? From the definition of the &function [Eq. (25)] one cLtn write
8
=
Y( V , l V a ) [ v ( p , ) ” 3 F ( p ~ ) l
(25a)
where the subscripts refer to viscosity and sedimentation measurement s. If either Ti, < V,(p, = p . ) or p , < p8(V,,= V , ) the experimental /3 could easily give a false axial ratio much lower than either p , or p , . For example, if the volumes are kept identical, but p , = 14 and p , = 18, we have then
814,is
=
8i4[F(pie.)/ F ( pi41 1
t
= 818 4p14) l Y ( P 1 8 ) i1/3 =
2.28 X lo6
which corresponds to an axial ratio of 6. Likewise, if p is kept constant but
VISCOSITY OF MACROMOLECULES
36 1
V , is smaller than V 8, a low &value will also result. This illustration by no means implies that fibrinogen must require two equivalent ellipsoids for viscosity and sedimentation. It is not clear why the p-function for fibrinogen behaves LLabnormally,”if the other weight of evidence is accepted as a correct description of the molecule. I t is even possible that the conclusion of Siege1 et al. (1953) may eventually turn out to be the correct one. There is need for more extensive tests for the @-function. As mentioned earlier, ideally the best answer should come from the determination of the &function. Unfortunately at present the rotary diffusion coefficient is usually the least reliable quant,ity in all hydrodynamic measurements because of errors inherent in the physical methods of flow birefringence and perhaps also non-Newtonian viscosity (see Section IV) . (Electric birefringence also may not give the same rotary diffusion coefficient as the other two methods, since the equivalent ellipsoids can be different under shearing stress and under electrical field.) Edsall (1954) has also illustrated the impossibility of evaluating the axial ratio from the 6function. The latter was about 0.80 for fibrinogen which corresponded to a prolate ellipsoid with an axial ratio of more than 300. If the rotary diffusion coefficient were only about 15% greater than that listed in Table V the calculated axial ratio would decrease to between ten and twenty. 4. Tobacco Mosaic Virus
The size and shape of tobacco mosaic virus has been the subject of numerous investigations both in solid state and in solution. From X-ray studies (Bernal and Fankuchen, 1941; Franklin and Klug, 1956; Caspar, 1956; Crick and Kendrew, 1957) and electron microscopy (Wilkins et al., 1950; Williams and Steere, 1951; Williams, 1954; Steere, 1957; Hall, 1958), it seems well established that the rodlike virus has a length of about 3000 A and a diameter close to 150 A. There is disagreement on the solution properties of the protein however, perhaps because of the difficulty in obt8ainingnearly monodisperse preparations. For example, the values of intrinsic viscosity from different laboratories were reported to vary from 0.25 to more than 0.6. In order to make any meaningful interpretations of the physical properties it is desirable to use the same preparation for all the measurements. Recently, Boedtker and Simmons ( 1958) have carefully reinvestigated this protein with a combination of light scattering, flow birefringence, viscosity, and sedimentation methods. Their results are summarized in Table V. The same preparation was also studied by Hall (1958) whose electron microscope measurements indicated that the number-average length was 3020 A and that more than 85 % of the 201 particles counted had lengths between 2800 and 3200 A. Thus the preparation was regarded as essentially uniform.
:G2
J E N TSI Y h N G
The evaluation of the equivalent ellipsoid after Scheraga and Mandel-
kerii ( 1 9 S ) led to @ = 2.61 x lo6 and 6 = 1.06. The former corresponded to L L ~ I:mid ratio of 18.5. If the experimental errors were considered to be
f 2 % , the axial ratios varied from 16 to 21, which fell into the rmge observed from the X-ray and electron micrograph studies. The &value gave an aria1 ratio of only 8. By assuming a f15 % error for the &value (about three times that for the calculated length) the axial ratio would vary from 5 to 20. On the other hand the length of the equivalent ellipsoid can be estimated with less ambiguity. As has been shown in Table 111 it varied from :3340 to 3550 A from flow birefringence to 3450 to 3710 A from intrinsic viscosity at zero gradient if the axial ratio was assumed to be 15 and 25. If one now turns to the conventional treatment by assigning an arbitrary water of hydration of 0,0.2, and 0.4 gm per grams of protein the corresponding lciigths of the hydrated protein are found to be 3680,3590, and 3540.4, respectively. These results compare very favorably with those mentioiied earlier, despite the fact that the use of hydration involves many debatable sssumpt ions. As Boedtker and Simmons (1958) have pointed out the equivalent length as obtaiiicd from the hydrodynamic measurements, about 3600 A, was appreciably larger than that from light scattering and electron microscope studies. The reliability of the Perrin equation to represent accurately a cylindrical particle has thus been questioned. Haltner and Zimm (1959) have measured the rotary frictional coefficient (kT/0) of carefully machined brass rods and prolate ellipsoids. The axial ratio of these models was so chosen that it approximated the actual ratio found in tobacco mosaic virus ( p = 20). According to these authors the reciprocal of the rotary diffusion coefficient for the rod (square ends) was 40% greater than that cdculated from the Burgers’ approximation for rods (1938) and the ratio of the coefficients between the ellipsoid and the rod was &/Or = 1.56. interesting discussion on the Burgers’ equation has recently been offered hy Broersma (1960) in his new theory on the same subject.] The latter finding seemed also to indicate the inadequacy of I’errin’s equation for rodlike particles. Yang (1961a) however has pointed out that the two models used in these experiments difkrent in volume, although they had the samc length and axial ratio. If one considers a hypothetical equivalent ellipsoid having the same volume and axial ratio as the rod, the ratio of the coefficients between the previous smaller ellipsoid and this equivalent ellipsoid is 1.50, which indeed agrees very well with Haltner and Zimm’s experimental value of 1.56. Thus the l’errin equation appears to be equally applicable for these “fat” rods, although the equivalent major and minor axes in this case were 14.5% greater than those of the rods. This fictitious equivalent ellipsoid is certainly by no means unique. Nevertheless it a t
VISCOSITY OF MACROMOLECULES
O(i3
least gives us a self-consistent approximate relationship between the rod and its equivalent ellipsoid. If the same argument can be applied to tobacco mosaic virus the length of the rodlike particle as deduced from the hydrodynamic measurements would have been 3600/1.145 or nkout :
A . Theories for Rigid Particles The theory of non-Newtonian viscosity for ellipsoidal particles was first explicitly stated by Kuhn and Kuhn (1945), using Peterlin’s distribution function (Peterlin, 1938) and Jeffery’s hydrodynamic treatment (.Jeffery, 1922-1923) [Eq. (lo)]. More elegant treatments have recently been developed by Saito (1951), using the same ellipsoidal model, and also by Kirkwood and his co-workers (Kirkwood, 1949; Kirkwood and Auer, 1951 ; Kirkwood and Plock, 1956; Riseman and Kirkwood, 1956) for rodlikc particles. The equivalence of the three theories has also been demonstrated by Saito and Sugita (1952). The general solution of Eq. (10) for the viscosity increment, v, can be expressed in the form v=a-bba2+ca4...
(30)
with the axial ratio, p , as a parameter. Here LY = G / @ and a, b, c, etc. are constants. The physical picture is also very simple. With increasing shearing stress the particles tend to orient more toward the streamlines and cause less energy dissipation than a t zern shearing stress; consequently, the viscosity drops. Precise numerical solutions of Saito’s equations ( 1951) have been computed by Scheraga (1955) in the manner employed previously for flow birefringence (Scheraga et al., 1951), using the Mark I computer. The values of v are tabulated as a function of p and LY (Table XII). The v values a t
364
J E N TSI YANG
a = 0 are identical with those listed in Table VIII according to the calou-
lations of Mehl et al. (1940). The ratios of v,/v,,o can also be converted to the experimental quantities, [q],/[q] ,=o , by the simple relationship
(37) (Table X I I I ) . Because of the limitations of the internal storage ettpacity of theMark I calculator these theoretical values are limited to a = 60. This range should be more than adequate for most of the experimental measurements. Empirically a plot of v or [ q ] , / [ q ] o E o versus a a t constant p on a double logarithmic scale was found to yield a straight line at a above five for both prolate and oblate ellipsoidal models (Yang, 1959). Thus the upper limit of a in the tables can be further extended by another order of magnitude or two through t)his simple extrapolation. Good agreement between the experimental results and the extrapolated theoretical values was recently reported. This procedure is arbitrary and without theoretical j ustification and therefore should be used with reservations.
B. Determination of Particle Length (or Diameter) The gradient dependence of viscosities of rigid particles leads to a new method for determining the particle shape. Like the flow birefringence technique (for recent reviews, see Cerf and Scheraga, 1952; Scheraga and Signer, 1960) it gives a direct measure of the rotary diffusion coefficient or the relaxation t>imeof the ellipsoids of revolution. For highly asymmetric particles of, say, p > 10 the ratio of [ q ] , / [ ~ ] ~ = oas a function of a( = G / e ) is very insensitive to the chosen axial ratio, p . Thus the calculations of particle length or diameter are essentially identical with those employed in the flow birefringence measurements. First, one has to choose either a prolate or an oblate model from other available information and then make a rough estimate of the axial ratio, for example, from the intrinsic viscosity a t zero gradient using the conventional treatment (with 8 ) .Next, from the experimental [q]o/[q]a=o or [q]r/[q]r=o values the corresponding a-values can be read from the Table XI11 in the Appendix, noting that a = G / O = (T/T)/(qoe/T)
(38)
(For detailed tabulations, see the original reference.) For axial ratios other than those listed the a-values can be obtained through interpolation or extrapolatlion, although this step is usually unnecessary for very large axial ratios. Thirdly, with a known for each chosen rate of shear or shearing stress, the quantity qo8/T can immediately be calculated according to Eq. (38). Finally, the particle length or diameter can be determined from Perrin's equations [Eqs. (19)]. If the calculated lengths or diameters vary with the gradient it is a clear indication of the degree of polydispersity. The
VISCOSITY OF MACROMOLECULES
365
dimensions as determined from the non-Newtonian viscosities again represent those of the equivalent hydrodynamic ellipsoids. The arguments raised in Section I11 can equally well be applied here. According to theory a rigid sphere does not have non-Newtonian viscosity and for axial ratios very close to unity the decrease in the intrinsic viscosities with increasing shearing stress is also very small. If in addition the dimensions of the particles are small (i.e., large q o B / T ) it is necessary to employ very high shearing stress so that a becomes large enough to be measurable. Thus for practical purpose this method is most useful only for highly asymmetric particles having very elongated or flattened dimensions which usually exhibit significant gradient dependence of their viscosities. To illustrate this point for protein solutions, consider the commonly used Ubbelohde or Ostwald type viscometers having a shearing stress of about 10 dynes cm-2 (or a rate of shear of about 1,000sec-’ for aqueous solutions). No significant non-Newtonian viscosity will be detected within experimental errors (a 1) at, say, 25°C for particles having a length (for prolate) less than 1900 to 2900 A or a diameter (for oblate) of about 1500 A or less (for axial ratios from 5 to 300). If the applied shearing stress is increased by tenfold the corresponding limits would become 9001300 A and about 700 A, respectively. Thus for most solutions of globular proteins the gradient dependence of viscosity would hardly be detected in ordinary capillary viscometers. On the other hand for highly elongated particles such as tobacco mosaic virus or collagen the gradient dependence could be quite appreciable when the shearing stress is in the order of 10 dynes cm-2.
C . Experimental Conjirmation The theory of Saito has been confirmed experimentally by measuring the gradient dependence of the viscosities of poly-7-benzyl-L-glutamates (in a-helical form) (Yang, 1958a, 1959). Following the rheologists’ practice, one can present the experimental data in a series of flow curves as shown in Fig. 4, where the rate of shear is plot,ted against the shearing stress on a double logarithmic scale. The viscosities appear to approach Newtonian (where the slope is unity) a t low shearing stress, followed by a marked drop above a critical shearing stress, 7 , , and approaching another Newtonian region a t very high shearing stress. The S-shape curve shows a more striking curvature with increasing concentration. From these flow curves the intrinsic viscosities at either constant shearing stress or constant rate of shear can be calculated. Yang has chosen the former on the basis of Eq. (38) which in turn is related to Eq. (37) hy
[~la/[~la=o
=
F’(7/T),,
rlo@/T = constant
( 378 1
366
JEN TSI YANG
for any particular polymer. Thus by plotting [&/[&o against r / T the polymer under study will yield a composite curve independent of the solvent viscosity and/or temperature employed, provided that no conformational change occurs under these conditions. In this respect it is noted that measurements in an Ostwald or Ubbelohde viscometer are made under virtually constant shearing stress (see Section V ) . The corresponding rates of shear are no longer constant under these circumstances and vary inversely with
5
4
2
1
0
1
3 4 5 log f . FIG.4. Typical flow curves of an a-helical polypeptide in m-cresol at 25°C. The experimental points at T > lo4dynes cm-2 were omitted in the plot. Concentrations: 0 , 0.379%; A, 0.502%; X, 0.621%; A , 0.776v0; 0 , 0.918%. Reproduced from Yang (1959). 2
the concentrations of the solutions, although by tradition most>workers still prefer to calculate the rate of shear for each solution and express the viscosities as a function of the latter. The agreement between the theory and experiments can test be illustrated in Fig. 5 , where the solid and broken lines were calculated on the basis of the known physical properties of the polymers. The obvious feature in the figure is the sharp drop in [q] wit,h increasing shearing stress which is in striking contrast with thc rather mild non-Newtonian behavior of the same polymer in its randomly coiled form (see below). This marked difference between rods and coils clearly provides a new means for the study of
367
VISCOSITY OF MACROMOLECULES
conformational changes in proteins, for example, protein denaturation. Also from the figure the non-Newtonian viscosity becomes significant a t a much higher shearing stress for the shorter particles than the more elongated ones, mainly because the former have a larger 7108/T [Eq. ( 3 8 ) ] .Furthermore, since each polymer has its characteristic viscosity curve the composite curve of many components in a polydisperse system would conceivably result in a broadening of the non-Newtonian region, as is indeed the case in Fig. 5 . Since the gradient dependence of the intrinsic viscosity of rigid particles gives a direct measure of the rotary diffusion coefficient it is possible in I
I
I
I
I
I
I
I
1.0
0.8 0
4
0.6
z 7 0.4 Y
Y
0.2 0
I
3
2 Log
4
7
FIG.5. Shearing stress dependence of the intrinsic viscosities of three a-helical polypeptides. The lines are theoretical curves (broken lines t o the right of the arrows being extrapolated theoretical curves): 1, q@/T = 0.76; 3, q&/T = 0.054; 2, calculated on the basis of three parts curve 1 and one part curve 3. Reproduced from Yang (1959).
principle to calculate the Scheraga-Mandelkern &function [Eq. (26)] from viscosity measurements alone, provided that the molecular weight is known from other physical methods. Here again the same uncertainty as discussed in Section I11 applies particularly when the particles are heterogeneous. Cerf (195813) has suggested that since the shape of the theoretical curve for [v],/[o],,o as a function of a is dependent on the model and also to some extent on the axial ratios, one can in principle determine both simply by superimposing the experimental [ ~ ] a / [ v ] o = versus o log G curve on the theoretical [~]u/[q],Eoversus log a curve for prolate or oblate ellipsoids. From the logarithmic scale one then calculates the rotary diffusion coefficient, since log a = log G - log 8.With p and 8 known the Perrin equation gives the value of the major and minor axes and thereby the volume. Furthermore with p (and thereby the viscosity increment, v) and V , deter-
368
JEN TSI YANG
mined, Eq. ( 2 2 ) in turn gives the molecular weight,. Thus a single intrinsic viscosity curve can provide data on both the size and shape of the particle. Cerf’s suggestion has not been tested but some formidable difficulties in practice can be foreseen. First the particles must be monodisperse and the experimental data of the highest precision. From the theoretical tables it is clear that the effect of axial rat,io on the gradient dependence of the viscosity becomes significant only when p is rather small, say, less than 10. Yet precisely in this range the intrinsic viscosity is usually small and its non-Newtonian behavior is difficult to detect. On tjhe other hand it is almost impossible to distinguish the theoretical curve for one axial ratio from t,he other if they are fairly large. To further complicate the problem any degree of polydispersity will distort the experimental curve so that no comparison can be attempted. Nevertheless it will be of interest to test this method if the particles are monodisperse and have low axial ratio arid also small rotary diffusion coefficient. The last requirement makes the measurements possible without resort to the use of high shearing stress.
D . Comparison with Flow Birefringence Method With the development of the non-Newtonian viscosit,y theories it is now possible to compare the rotary diffusion coefficient and thereby the calculated length (or diameter) of the rigid particles as obtained from this technique with that from the commonly used flow birefringence method. Since both measurements depend upon the same molecular distribution funct,iori (Section 111) they should give an identical measure of the rotary diffusion coefficient. Differences, however, will arise if the system under study is heterogeneous. The mean intrinsic viscosity is calculated from Eq. (7) whereas the mean extinction angle, x , for flow birefringence is defined by the Sadron equation (1938) : tan 2x
=
Z A n i sin 2 x i / C A n ; cos 2xi
(39)
where the subscript i refers to the ith component which, if present alone in the solut,ion, would give an extinction angle, x i , and a birefringence, Ani. Consequently the mean length as calculated from both methods will not only vary with the applied shearing stress but also depend on the equation used for such calculations. This can best be illustrated by the recent results on tobacoo mosaic virus (Yang, 1961a) (Fig. 6 ) . The flow birefringence average length decreases, as expected, montonically with increasing shearing stress, since the orientation of the particles is heavily weighted by the longer ones a t lower shearing stress. The viscosity-average length however reveals a maximum for the following reason. At zero gradient the mean length can be calculated from Eq. (27b), which is usually closer to a weight average. As soon as the shearing stdress increases, however, the
369
VISCOSITY OF MACROMOLECULES
longer particles are first oriented, resulting in a drop in viscosity and thereby a decrease in the mean rotary diffusion coefficient which in turn causes an increase in the calculated mean length. At still higher shearing stress even the shorter particles begin to be oriented and as a consequence the mean rotary diffusion coefficient gradually increases and the mean length decreases again. Once all the particles are oriented parallel to the streamlines (at infinite shearing stress), there will be no further drop in viscosity and the mean length probably approaches the same average as that a t zero gradient. On the other hand the flow birefringence averages are quite complicated and still not well known. Goldstein and Reichmann (1954) have shown that as shearing stress approaches zero the flow birefringence average 3 1/3 length, ( L ) = ( ( L 6 ) / ( L)) which is even more heavily weighted than the z-average. The same authors have also suggested a number average when
-
Boo0
T
FLOW 8/REFR/N66NCE V/SCOS/TY
6@30 *J
3
$4000
2000
o
10 ao 30 40 so SHEARING STRESS, f dyner cm-'
1
FIG.6. The hydrodynamic lengths of a tobacco mosaic virus sample. Reproduced from Yang (1961a).
the shearing stress becomes infinite. This perhaps explains the crossover of the two curves in Fig. 6. It is clear that the flow birefringence technique is extremely sensitive to the degree of polydispersity, much more so than the viscosity method. At very high shearing stress both curves in Fig. 6 appear to level off gradually. This merely reconfirms the fact that any apparently constant length as calculated over a narrow range of shear can be quit,e misleading. On the other hand for flow birefringence the extrapolated length (to zero gradient,) may approach the upper limit of the longest particles but definitely does not represent the mean length a t zero gradient>.
E. Theories for Flexible Coils The gradient dependence of viscosity of flexible coils is a much more complicated problem than that of rigid particles. I n addition to orientation under shearing stress the permeable and deformable nature of these molecules leads to such theoretical and conceptual difficulties that no adequate theory has as yet been found to be of general applicability. Kirkwood and
370
J E N TSI YANG
his co-workers (Kirkwood and Riseman, 1948; Kirkwood, 1949, 1954; Riseman and Kirkwood, 1956) first developed a rigorous theory using a general statistical model with hydrodynamic interaction. Since the gradient dependence of molecular conformation is not known explicitly for random coils these authors have considered only the undeformed equilibrium conformation, thus leading to Newtonian viscosity only. Rouse (1953) and Zimm (1956) employed two somewhat different models, elastically connected beads without and with hydrodynamic interaction, respectively, but also found no gradient dependence a t all. This conclusion is not in accord with existing experimental evidence. It, is noted that the three theories are derived by the use of a perfectly flexible Gaussian coil model, which does not fit any real polymer chain. Earlier this idea of imperfect flexibility led Kuhn and Kuhn (1’346) to introduce the concept of “internal viscosity,” which has been further extended by Cerf in a series of papers (1951, 1955, 1957, 1958a, 1959). In either case the net effect is to stiffen the molecular chain and both treatments Iead to a gradient dependence. On the other hand, by considering the effect of anisotropy of the polymer conformation on hydrodynamic interaction rather than internal viscosity Peterlin and CopiE (1956) and Ikeda (1957) reach essentially the same conclusion as Kuhn and Kuhn and Cerf. The results of these four theories can all be expressed in a series expansion of the intrinsic viscosity in powers of the rate of shear: [ ~ ] ~ / [ & = o = 1 - constant
+
(M[.rl]u=o.rloG)2
(40)
alt,hough the numerical constants are not identical with one another, At present there is no unanimity of opinion among those who hold various theories concerning the origin of the gradient dependence of viscosity. It may also be mentioned that Bueche ( 1954), using the same model as Rouse and Zimm, developed a theory which predicts a drastic drop in intrinsic viscosity with increasing shearing stress in the same manner as that for rigid particles. This surprising contradiction between Bueche’s and Zimm’s theories has been discussed by Peterlin and CopiE (1956) who suspect that Bueche’s conclusion probably arises from an incorrect treatment of hydrodynamic interaction. Very recently, Zimm (1960) has given a more exact treatment of the hydrodynamic interaction than in his earlier work. This refined theory also leads to a gradient dependence. Experimental evidences of the non-Newtonian viscosities of polymer chains are too numerous to list here. Most experimental and theoretical results can be described by a general equation: [qIG =
[qjc=o(l - constant G ” )
(41 )
It has the same inverse8 shape as that found for rigid particles (Fig. 5 )
VISCOSITY OF MACROMOLECULES
371
although the extent of drop in viscosity is usually much milder than in the latter case. Many factors influence the constant in Eq. (41) and all experiments are consistent in demonstrating that the effect increases sharply with molecular weight [see Eq. (40)]. Of equal importance is the effect of solvent, the gradient dependence of viscosities of any polymer being much more pronounced in good solvents than in poor ones (Sharman et al., 1953). It does not disappear, however, even in an “ideal” solvent, i.e., at Flory’s 0-temperature (Flory, 1953), although the effect is much reduced under these conditions (Passaglia et al., 1960). At 0-temperature the root-meansquare end-to-end distance, (ri)1’2,of any real polymer chain is still consistently greater than that calculated on the assumption of completely free rotation about each chemical bond, (T~,,,)~”, and thus the molecular conformation is not quite equivalent to a perfect Gaussian coil. On the other hand, for polymers of different chemical compositions having different degrees of flexibility (or stiffness) the gradient dependence of viscosities is reported to be much more pronounced, the more extended the chain conformations (Passaglia et al., 1960). In the extreme case where the polymer chains are completely stiff and fully extended they resemble rigid particles, for which both theory and experiments are in complete agreement. These observations are interrelated and a general pattern emerges, that is, the non-Kewtonian viscosity of polymers is predominantly determined by their molecular conformations. If the polymer chains are coiled up through the use of a poor solvent they invariably exhibit little gradient dependence of viscosity. Conversely, for very stiff chains or for those which are highly extended because of strong solute-solvent interaction in a good solvent, a pronounced gradient dependence of viscosity usually results. Empirically this dependence has been found to disappear only when experimental data in various solvents are extrapolated to the ideal conditions under which the polymer chains have the conformation dictated by the hypothetical completely free rotations about each chemical bond (Passaglia et al., 1960). Viscosity theories for flexible coils are not directly applicable to most proteins. Qualitatively, the fact that flexible polymers have a less marked non-Newtonian viscosity than the rigid particles should be of interest in the studies of protein denaturation. This is further illustrated in Fig. 7, representing the viscosities of a poly-y-benzyl-L-glutamate in both the a-helical and randomly coiled forms (Yang, 1958a). The striking drop in viscosity for the rods is a direct contrast to the small gradient dependence for the coils. In this particular case the curves for the two forms at comparable concentrations cross each other. One is therefore led to believe that the a-helices remain stable even when subjected to very high shearing stress. Strong evidence against any conformational change of the helices under shearing stress comes also from the close agreement between theory
372
J E N TSI YANG
and experiments for these helices over the entire range of shearing shress studied (see Fig. 5 ) . From the findings in Fig. 7 one may also deduce that most denatured proteins, if present as very flexible coils, will exhibit little or no non-Newtonian viscosity under normal experimental conditions.
F . Power Law of Viscosity The value of the exponent, n in Eq. (41) has been the source of considerable controversy among the rheologists: t,he so-called power law of vis-
8
ea
6
I-?\\ !-
\ ,.~m
-Cresal
R
4
2
Dichloroocrtic
0
1
3
2
4
5
Lop 7
FIG.7. Corn srison of the non-Newtonian viscosities of a polypeptide in helical (in m-cresol) and randomly coiled (in dichloroacetic acid) forms. Reproduced from Yang (195th).
cosity. By simple geometric analysis viscosity should be an even-function of shearing stress or rate of shear independent of the direction of flow; that is, only when the exponent n is an even number can the numerical value of viscosity remain unchanged by reversing the sign of T or G from positive to negative. Indeed most theoretical treatments predict a value of 2 for n. A notable exception is Bueche’s theory (1954) which reaches values of 2 and $5 for free-draining and nonfree-draining coils respectively. It is noted however that the agreement with experiment which Bueche demonstrates
VISCOSITY OF MACROMOLECULES
373
could equally well be achieved with other functions and the curves he presents are by no means unambiguous on this point. Experimentally most published data for polymer coils suggest an odd-function, i.e., n = I, as T or G approaches zero (for a brief review, see Hermans, 1957). To obtain the value of this exponent involves extrapolation to zero gradient and this is often difficult. For rigid particles both theories and experiments are in accord with an even function (see Section IV, C) . If the macromolecular system is polydisperse i t is possible to obtain a pseudo-odd function even though each component obeys an even-power equation (Yang, 1959). Very recently Eisenberg (1957) has shown that even for monodisperse rigid particles a linear approximation can be obtained over certain limited ranges of rate of shear. By examining a theoretical curve for rigid particles having a particular rotary diffusion coefficient he has illustrated that the correct limiting quadratic law in this special case was [v]a/[q]o=o =
1 - 3.4 X 10PG2
for G < 140 sec-'. If a term in G' was added to the above equation the range of linear extrapolation could be extended to G < 280 sec-'. Unless [qlo=O was known from measurements a t G < 30 sec-' in this particular case several incorrect empirical equations could be found to fit the theoretical curve within different ranges of rate of shear. For example, [q]a/[q]o=o =
between G
=
1.065 - 9.1 X 10-4G
100-400 sec-I, and [~Ia/[q]u=o = 1/(0.2
+ 5.97 X 10-"G''2)
over a wide range of rate of shear provided that G > 300 sec-'. Although Eisenberg only discussed the intrinsic viscosity of rigid particles the same argument is expected to apply to flexible coils as well. Thus the power-law controversy appears to have been overemphasized. At least for polymers which are not well fractionated the effect of polydispersity can complicate the interpretation of the power law.
G. Complex Viscosity The viscosity of a macromolecular solution can undergo changes when subjected to a periodic shear wave of frequency, w , instead of a steadystate shearing stress. The response of the particles to such a sinusoidally oscillating shear can be expressed in terms of a complex viscosity, q*: ?I* = 7]R
-
$7,
(42)
where the subscripts R and I refer to the real and imaginary parts of a
374
JEN TSI YANG
complex number and i = 4-1.The real part is the viscosity and the imaginary part is the complex modulus of elasticity of the solution. I n steady-state flow where w = 0, the q1 term, together with the components of the complex modulus of elasticity, vanishes and the real part in Eq. (42) simply becomes the steady-state viscosity. By plotting 7~ as a function of the frequencies, w , on a logarithmic scale the values of 7~ fall from a low a-plateau to another plateau at very high frequencies, in the same manner as the non-Newtonian behavior of the solution varies as a function of the shearing stress. The physical picture is as follows: a t low frequency the particles are oriented (and also extended for flexible coils) by the applied shearing wave, but a t the same time they rotate in the flow gradient. The input energy is thus gradually dissipated and a true viscosity appears. At high frequencies, however, the particles are oriented (and extended for flexible coils) only slightly in one phase of the motion which immediately reverses itself. Thus the stored energy in the particles does not have time to dissipate but returns to the fluid. Accordingly there is very little loss in energy and the viscosit*ybecomes small. Since the complex viscosity is concentration-dependent just in the same manner as the steady-state viscosity, one can define a complex viscosity increment, v*, and a complex intrinsic viscosity, [7]*,similar to that for steady-state viscosity v* =
VR
- ivI
( 42%)
-
( 4% )
and
[?I*
= [7lR
if711
For rigid particles the frequency dependence of the real part in Eq. (42a), V R , has been solved by Cerf (1952) and can be written as
For ellipsoids of revolution the numerical values of v A and V B have been tabulated by Scheraga (1955), and the sum of v A and V B (i.e., V R a t w = 0 ) is identical with the viscosity increment from Simha’s equation. Thus Eq. (43) provides an alternative method to that of the non-Newtonian viscosity for the determination of the rotary diffusion coefficient, 0. Cerf has also pointed out that 8 is determinable from the slope a t the inflection point (1.P.) of the vR versus w-curve, i.e., w(1.P.) = 2 d 3 0 . At present, however, no experimental test of Eq. (43) has as yet been reported. Several theoretical treatments of complex viscosities of flexible polymers have recently been developed among which Rouse’s theory (1953) has received wide attention. The general agreement between the theory and ex-
VISCOSITY OF MACROMOLECULES
375
periments (Rouse and Sittel, 1953) is very good, although there appears to exist a systematic difference amounting to about 50% between the calculated relaxation time and the experimental one. This discrepancy is small in view of the wide range of molecular weights employed for various polymers. This subject is of great interest to the rheologists but as yet it has not been applied to most proteins.
V. EXPERIMENTAL METHODS Viscometry has become such a routine technique in many laboratories that it seems unnecessary to mention the usual precautions such as the clarification of sample, the cleanliness of the viscometer, the temperature control (to 0.01"C or better), the timing device, the vertical alignment of capillary viscometer, etc. In this section we will only describe the basic equations used in the viscosity measurements and various corrections and precautions which are necessary to insure reliable results. (F'or a monograph, see, for example, Barr, 1931.)
A. Types of Viscometers Viscometers include the capillary, coaxial cylindrical, cone-and-plate, falling-ball type, etc. The capillary and, to a lesser extent but of increasing importance, the coaxial cylindrical viscometers, in numerous modifications, are the two most commonly used in scientific laboratories. 1. Capillary Viscometers
The flow in a capillary is inhomogeneous in the sense that the shearing stress, r , and the rate of shear, G, vary with the position of the fluid inside the capillary. The velocity of the flow is maximum along the central axis but gradually drops to zero at the wall, whereas the reverse is true for the shearing stress and rate of shear. For a Newtonian flow the viscosity, q( = r / G ) , remains constant at any point inside the capillary even though both r and G vary considerably from one point to another. On the other hand, for a non-Newtonian flow the viscosity varies along the radial distance of the axis. a. With Essentially Constant Pressure Head. For a Newtonian flow the viscosity can be calculated by the following equations: T,,,
=
AP*R/BL
(44a)
G
=
4Q/?rR3= 4V/lrR3t
(44b)
lrR4AP.t/8LV
(44c)
q =
Here T~ is the maximum shearing stress at the wall, G the nominal rate of shear, AP the applied pressure, Q( = V / t ) the volume flow rate, and R
376
JEN TSf YANQ
the radius and L the length of the capillary. Equation (44c) is the wellknown Hagen-Poiseuille equation. [For the derivation of Eq. (44c), consult any st,andard textbook on hydrodynamics.] In an Ostwald or Ubbelohde viscometer where the hydrostatic pressure can be represented by A h . p . g , Eq. (44c) becomes 7 =
nR4pgAh*t/8LV
( 44d )
Here Ah is the pressure head, p the density of the solution, and g the acceleration of gravity. Strictly speaking, Ah in this type of viscometer varies during the measurement. It is not correct merely to take the average Ah at the beginning and ending of the measurement since the flow is faster a t first than near the end. The average Ah can however be calculated by the Meissner equation Ah
=
(Ah1 - Ahz)/log, ( A h i / A b )
(45)
where Ah1 and Ah2 are the hydrostatic heads at the beginning and ending of the measurement, (see Barr, 1935). The Hagen-Poiseuille equation is no longer directly applicable if a flow is non-Newtonian. It can be easily shown that instead of Eq. (44b) t,he volume flow rate in this case becomes (Hermans, 1959)
By st,raightforward differentiation one obtains 77, =
aR37kd7rn/d(Q T ~ )
and the apparent, experimental rate of shear, G, (44b) should then be corrected by the relation G(corr.)
=
G,(n
, as calculated
+ 3)/4
(46n)
from Eq.
(47P
where n is defined as the slope of the flow curve, i.e., dlog G,/dlog rrna t any chosen T~ or G, . Alternately, the apparent viscosity, 7, , as calculated from Eq. (44c) can be corrected by the relation vT,
(corr.)
=
Va/(
1 - @logsa/dlog7rn)
(48)
(see also, Krieger and Maron, 1952). Several other modified forms of Eq. Equation (47) has been derived previously by the rheologists from the so-called power law G = k7. which assumes a constant n a t any chosen applied pressure ( k being a constant). This derivation is not exact since n varies with the position of the fluid inside the capillary. It is unity a t the center of the capillary where the flow is Newtonian and becomes greater than one toward the wall when the flow is non-Newtonian. Equation (47) can now he derived without this unrealistic assumption,
VISCOSITY OF MACROMOLECULES
377
(48) have been reported in the literature which are essentially identical to one another. Since we are more interested in the correction to intrinsic viscosity due to inhomogeneous flow in a capillary it can be further shown that [ql,,,, (con-.
1
=
+ td[qlO/dloge.r, [91a(1 + tdlog[qla/dlogr,)
[TI,
=
(49)
where [q], is the uncorrected, apparent intrinsic viscosity a t shearing stress, r m , as customarily calculated from the flow times of the solutions and solvent. For Newtonian flow where n = 1 no correction for the nominal rate of shear [Eq. (44b)l is necessary. This is also obvious from Eqs. (48) and (49) where q, and [q], are independent of the applied shearing stress when the flow is Newtonian and therefore their derivatives become zero. On the other hand the n values could be as high as 3 or 4 for concentrated solution in the non-Newtonian region (see Fig. 4). For most viscosity measurements where the solution viscosity may be only several times that of the solvent this correction to n appears to be insignificant. Nevertheless such precautions should by no means be overlooked since any small errors in the absolute viscosity could be enlarged several times in the specific viscosity. Indeed according to Eq. (49) the errors are by no means negligible even if all the viscosities are measured in very dilute solutions. Precise graphical determination of n, dlogq./dlogr, , d[&/dlog,r, , or dlog[q],/ dlogr, is rather difficult. An improved procedure which can be adapted to Eqs. (48) and (49) has recently been suggested by Maron and Belner (1955), which however still requires some guesswork. For very precise calculations a computer program can also be set up to resolve this difficulty. In the past the Kroepelin equations (1929) for the mean rate of shear
G
=
8V/3?rR3t
and the mean shearing stress 5 = AP*R/3L
( 50b 1
were widely used. These equations are derived oiily for a Newtonian flow and there seems no justification to apply them to a non-Newtonian flow (see also, Goldberg and FUOSS, 1954). If one is interested only in the extrapolation of the viscosities to zero gradient it is immaterial whether Eqs. (44a and b ) or the Kroepelin equations are employed. On the other hand with [q]a/[q]a-o as a function of a the calculated rotary diffusion coefficient, 8 , will differ by a factor of y$ from the two sets of equations. However Eqs. (44a and b ) and (47) are preferred to the Kroepelin equations, since the former are derived without any assumptions.
378
JEN TSI YANG
b. With Continuously Varying Pressure Head. Instead of multibulbs, a wide precision-bore tubing can be attached to the capillary of an Ubbelohde viscometer which constitutes a suspension type with a continuously varying pressure head when filled with the solution or solvent to be measured. Thus the volume flow rate in this case becomes Q = dV/dt instead of V / t . Again by straightforward differentiation the viscosities can be calculated from the following equation (Hermans and Hermans, 1958) : 1/vrm = - ( 2LS/r@R4)[4dlogeAh/dt
+
( dt/dlogeAh)d210geAh/dt2] (51)
where Ah is the pressure head a t time t, S is the cross-section area of the wide precision-bore t,ubing, and the other symbols have been defined previously. For a Newtonian liquid log,Ah is proportional to the flow time t and the second term in the bracket drops out. Thus a plot of log,Ah versus t yields a straight line and the viscosity can be calculated from the slope. For a non-Newtonian liquid the -dlog, Ah/dt decreases with decreasing Ah, since the viscosity is higher at lower pressure head. Thus the log,Ah versus t plot will show an upward curvature as the time t increases. I n most cases in particular with very dilute solutions, the second term in the bracket of Eq. (51) can usually be omitted without the introduction of significant errors. For the relative viscosity a t any chosen pressure head one can simply write as a first approximation vrel
=
(dlogeAh/dt)mlvent/(dlogeAh/dt)mlution
(52)
Precise graphic determination of the differential dlog,Ah/dt is again rather difficult when the flow is non-Newtonian. Equation (51) can equally well be applied to a U-tube type viscometer, in which a capillary is connected to essentially a manometer (see, for example, Maron and Relner, 1955). The only modification is that the pressure head, Ah, is calculated from the difference in the decreasing meniscus of the capillary arm and that arising in the manometer 5rm. 2. Coaxial Cylindrical (Rotational) Viscometer I n a rotational viscometer the solution is filled in the annulus between two concentric cylinders of which either the external ( Couette-Hatschek type) or the internal (Searle type) cylinder rotates and the other, which is connected to a torsion-measuring device, is kept in position. Let Ri and Robe the radii of the inner and outer cylinders, h the height of the cylinder which is immersed in the solution or its equivalent height, if end effects are present, w the angular velocity of the rotating cylinder, and T the torque (or moment of force) required to keep the velocity constant against the viscous resistance of the solution. It can be shown that the shearing stress (see, for example, Iteiner, 1960): r m = T/2*R2h (53a)
VISCOSITY OF MACROMOLECULES
379
the rate of shear4:
and the viscosity: 9 =
T(R%- R:>/4xhRTRfw
(53c)
Here R refers to Ri or R, if the inner or outer cylinder rot,ates. Just as in a capillary flow the rate of shear in Eq. (53b) for a non-Newtonian flow can be further corrected by an equation (see, Reiner, 1960) G(corr.)
=
G,.n(l - a ) / ( l - a”)
(544
where n is again equal to dlogG/dlog.r and a = (Ri/R,)‘. The rotational viscometer has an obvious advantage over the capillary one, due to the fact that the rate of shear across the annular gap is nearly constant and approaches constancy as the ratio of the gap to the radius of the cylinder becomes smaller. At the bottom of the inner cylinder the rate of shear is quite different from that across the gap, thus introducing some uncertainty into the measurements. This end-effect problem can be minimized or eliminated. An excellent discussion has been given by Mooney and Ewart (1934). The range of usage and accuracy of this type of viscometer is limited only by the response of the torsion-measuring device. I t can be so designed as to cover extremely low range of shearing stress. Unlike the capillary viscometer the construction of a rotational viscometer of high precision is a delicate problem.
B. Correctionsfor a Capillary Viscometer 1. Kinetic Energy
The derivation of Eq. (44c) assumes no acceleration along the axis of the capillary. This is not true at both ends of the capillary where the rate of flow is much faster inside the tube than outside. Thus the pressure drop, AP, is not caused entirely by the viscous resistance of the fluid and a portion of it is attributed to the velocity of the flow according to the well4The velocity of the rotating cylinder, u, equals the radius, r, times w . Thus, du/dr = rdw/dr O . The term on the left aide of the equation is the velocity gradient and the first term on the right is the rate of shear. Strictly speaking, the two terms in this case differ by a quantity, w . 6 Equation (54) is derived from the so-called power law of viscosity which, strictly speaking, is not exact (see footnote 3 on p. 376). According t o Mooney (1931) a general solution for a non-Newtonian flow in a rotational viscometer is not possible. Recently Krieger and Maron (1952) and Krieger and Elrod (1953) have proposed an approximate solution using Euler and Maclaurin’s method.
+
380
JEN TSI YANB
known Bernoulli theorem. From energy considerations (per unit time) one can write AP(effective) .Q = AP(expt1.) .Q -
1
Ql - p d Q d 0 2
(55)
The integral is the kinetic energy correction where p is the density, Q = V/t the volume flow rate of the fluid, and u the velocity of the flow a t point r from the axis of the capillary. For Newtonian liquids it can be shown that the integral term becomes pV3/n2R4t3, noting that dQ = 2nrdr.u, -du/dr = G T / T , = (4V/nR42)r, and u = (2V/rR4t)(R2 - r 2 ) .Usually an empirical constant m is introduced to correct the nonideality of the construction of the visrometer. Thus we have T(corr.) = AP(R/BL)
- mpV2/2r2R3Lt2
(%a)
and q(corr.)
=
nR4.AP.t/8LV - mpV/8nLt
(55h)
TCquation (55b) is frequently called the Hagenbach correction. The value of m is found to be of the order of unity; many workers prefer a value of 1.12, although there have been many arguments about its correct value. The best procedure seems to determine it experimentally. The above correction is derived for a Newtonian liquid and therefore becomes somewhat uncertain if the flow is non-Newtonian. According to Eq. (55b) the kinetic energy correction can be minimized by increasing the flow time per unit volume (i.e., t / V ) or the length, L, of the capillary or both. Practical consideration however limits the feasible range of the length. Increasing the flow time by using a larger volume of the solution also does not help a t all. The best method is to reduce the capillary radius since the flow time is inversely proportional to the fourth power of the radius. On the other hand if the capillary is too fine cleaning of the capillary will become a problem. The kinetic energy correction is also obviously smaller if the density of the solution is lower. 2. End Effect
A flow undergoes sudden contraction and expansion upon entering and leaving a capillary. Thus the velocity of the flow is less near the ends than that in the middle point of the capillary. This effect may be considered as equivalent to an increase in the effect,ive length of the capillary. It is frequently called the Couette correction. The Hagen-Poiseuille equation corrected for both the end and kinetic energy effects, can then be written as : t = nR4AP.t/8(L nR)V - mpV/8n(L nR)t (50)
+
+
VISCOSITY OF MACROMOLECULES
38 1
Here nR is usually of the order of several times the radius, R. Thus this correction becomes insignificant if the ratio of L / R is very high, say, over 50. On the other hand in the non-Newtonian viscosity measurements where the length of the capillary may be reduced c.onsiderahly in order to cover a wide range of shearing stress this factor may become appreciable. For an entirely different reason the rheologists have studied the effect of L / R on the apparent viscosity by deliberately reducing it to close to unity. By so doing they are able to determine the so-called recoverable shear which is one of the important properties of the viscoelasticity (Philippoff and Gaskins, 1958). This aspect is beyond the scope of the present review. [For experimental determination of the constants m and n in Eq. (56), see an excellent paper by Swindells et aZ. (1952).] Equation (56) is more commonly written as 7] =
at - @ / t
(56a)
To determine the constants (Y and @ one measures the flow time of two liquids, or one liquid at, two different temperatures, of kiiowri viscosities and d v e s the two simultaneous equations. Or, if one can vary the applied pressure then Eq. (5Ga) can be rewritten as g =
a'AP*t - a/t
(56b)
By plotting AP.t against l / t or APet' against, t one can determine the viscosity from the intercept or the slope of the straight line respectively. The experimental a- and &values should be comparable t,o those calculated from the dimensions of the capillary.
3. Anomalous Flow Near a Wall The distribution of particle orientations near a capillary wall is restricted hy the presence of such a rigid body (the wall) which introduces a preferred direction in an otherwise isotropic medium. As a consequence the rate of shear near the wall may not be a function of the shearing stress alone and there may be an effective velocity of slip a t the wall. This complication however is usually ignored in routine work. 6 Strictly speaking, t,he (Y- and 8-values in an Ostwald or Ubbelohde viscometer remain constant only if the liquids to be measured have the same densities. If this is not the case Eqs. (56s) and (56b) can be rewritten as:
(I
= ffPt
-8Pb
and q = a'AP+t
- pp/t
382
JEN TSI YANG
4 . Surface Tension and Drainage Errors
Correctlion to the pressure head due to surface tension can in principle be determined from the curvatures of the surfaces at both the upper and lower meniscuses inside the viscometer: AP8
=
2y/r
(57)
where y is the surface tension, r the radius of curvature, and APa the pressure head due to capillary rise. Since this effect occurs a t both meniscuses it cancels out provided that the two radii of curvature are identical. The drainage error arises from the fact that a small amount of solution or solvent adheres to the wall of the reservoir of a capillary viscometer during measurements. According to a theory by Zweegman, Tuijnman, and Hermans as quoted by Tuijnman and Hermans (1957) the fractional loss of the volume, - AV/V, to the wall for a Ubbelohde viscometer having a radius R, for the reservoir (assumed cylindrical) can be written as
- AV/V
=
(4/3R,) ( &/aRipg)l’’
( 5% 1
which in combination with the Hagen-Poiseuille equation [Eq. (44c)l becomes - AV/V =
(4/3R,) (AP. R4/8LR?pg)’I2
( 5%1
0.471Ah”2*R2/L’‘2* Ri
(58c1
or
- AV/V
=
noting that AP = Ah.p.g(Ah = pressure head, p = density of the liquid, and g = acceleration of gravity). Thus the drainage error increases with increasing pressure head and radius of the capillary, and with decreasing reservoir radius and length of the capillary. The drainage correction applies to both the solution and solvent and therefore becomes relatively insignificant when only the relative viscosities are considered. For practical purposes both surface tension and drainage corrections can be approximately incorporated into the constants in Eqs. (56a and b ) . 5 . Turbulent Flow
The viscosity as calculated according to Eq. ( 4 4 ) is meaningful only if the flow is laminar. For a capillary flow the rate of flow should not exceed a critical velocity which can be determined from its Reynolds number
(RN): R N = DUP/T
(59)
where D is the diameter of the capillary, u the velocity of the flow, p the density, and the viscosity of the fluid. The critical Reynolds number for
VISCOSITY OF MACROMOLECULES
383
most liquids has been found to lie in the range of 2,000 to 4,000. The situation is more complicated in a non-Newtonian flow where the viscosity varies with the shearing stress and thereby with the velocity of the flow. Various criteria for the onset of turbulence have been suggested but so far no common agreement seems to have been reached on this subject. One reasonable suggestion is to use the viscosity at the applied shearing stress rather than at zero shearing stress, although many other relations have also been reported in the literature. Equation (59) can be rearranged into
RN = 2Vp/rRqt
= 2Qp/rRt
( 59a 1
For aqueous solutions, say, at 25°C the lower limit of the critical Reynolds number will not be reached as long as V/Rt is well below 30. Thus for all practical purposes this complication will not arise in routine viscosity measurements. On the other hand if the non-Newtonian viscosity measurements are extended to a very high range of the shearing stress, it is desirable to check the possibility of this effect. 6. Density
The densities of a dilute solution and solvent are usually assumed to be almost identical and as such this correction was thought to be negligible in an Ostwald or Ubbelohde viscometer. According to Eq. (44c) the relative viscosity is ( 60a 1
d t o = Pt/POtO
Thus the ratio of flow times is actually a measure of the kinematic viscosity, v (see Table 11). tho = ( ? / P > / ( t l o / P o )
= v/vo
( “31
(The symbol v as used in this section should not be confused with that for the viscosity increment.) To obtain the intrinsic viscosity
one must in principle know the accurate values of densities a t different concentrations. This procedure is certainly too laborious for routine work. By defining an intrinsic kinematic viscosity
Tanford (1055), however, has shown that
384
JEN TSI YANG
v
where is the partial specific volume of the solute. This simple relation permits the determination of the intrinsic viscosity with ease. As an illustration Tanford has given the data on bovine serum albumin a t 25°C in 0.01 M KC1: [v] = 0.03436, P = 0.734, and po = 0.9975, which in turii yields [T] = 0.03704 [the last term in Eq. (62) being 0.00268], An accuracy of fO.O1 for 7 is sufficient for routine calculation since experimental data usually do not warrant the use of four significant figures for the intrinsic viscosity. This density correction may amount to several per cents for those globular proteins having low intrinsic viscosities. It becomes negligible if the intrinsic viscosities are very high.
C. Extrapolation to Zero Rate of Shear It has been a common practice to measure the viscosities in a multigradient viscometer. The relative viscosities a t each concentration are then plotted against the rates of shear, followed by extrapolation to zero rate of shear and the intrinsic viscosity is calculated from these intercepts for various concentrations in the customary Huggins plot. From both theoretical and experimental considerations, however, the shearing stress rather than the rate of shear should be preferred, although tradition has emphasized the latter (Section IV, C). I n view of the discussions on the power-law controversy (Section IV, F ) one may argue that extrapolation to zero rate of shear should be done by plotting the viscosities against the squares of the rates of shear. It can further be shown by simple differentiation that an equation such as Eq. (40) gives a zero slope at zero rate of shear in a q-G plot (i.e., the viscosities level off as G approaches zero). Thus a linear extrapolation will lead to a viscosity higher than its true value, although normal experimental errors usually make it difficult to detect such subtle differences. The situation is complicated further by the problem of polydispersity since the gradient dependence of the viscosities is less sharp for a polydisperse system than for monodisperse particles. It may not be difficult to fit the experimental data with a linear plot over almost any narrow range of the shear employed, thus giving a completely erroneous extrapolated value. The situation will be much worse if such a plot exhibits an upward curvature as the gradient approaches zero, since in this case the intercept on the viscosity axis depends entirely on how one draws a smooth curve through the experimental points and arbitrarily extrapolates them to zero gradient. The use of a semilogarithmic scale or some other relations may minimize the curvature, but this could be only illusory and the uncertainty of the extrapolated value still exists. All these considerations warn against any indiscriminate use of a linear approximation, unless one is certain that the range of the applied shearing stress is sufficiently low to warrant this pro-
VISCOSITY OF MACROMOLECULES
885
cedure. For rigid particles some information about their shape would immediately reveal the critical shearing stress above which the non-Newtonian behavior becomes significant.
D. Design of a Capillary Viscometer Many types of capillary viscometers have been described. Here we will discuss only some general considerations. According to Eq. (44a) one can construct a multigradient Ubbelohde-type viscometer by adjusting the length, L , the radius, R, of the capillary, or the applied pressure head, A P , or a combination of the three. The shearing stress can be lowered by increasing the length or reducing the radius. The latker however is limited hy the practical range of the flow time, and also the problem of cleaning (to remove dust, etc.). Since according to Eq. (44b) the flow time varies inversely as the fourth power of the radius, a reduction of the radius to one-half results in a sixteenfold increase in the flow time. It is also impractJical to compensate for this vast variation by reducing the volume of the flow. If the liquid flows by gravity, g, then A P = Ah'pvg. Thus the applied shearing stress can also be lowered by using a shorter Ah. There is however a lower limit of Ah beyond which appreciable errors will be introduced by the effects of surface tension, etc. To construct a high gradient viscometer one reverses the procedure by using a shorter length of the capillary, or by using a higher pressure head by either increasing the Ah or applying an external pressure with compressed (dry) nitrogen, etc. The use of a larger radius, however, is undesirable in this case, since it usually makes the rate of flow too fast. For very short length one has to correct for the kinetic energy and end effects. As an illustration the dimensions of three hypothetical Ubbelohde-type viscometers are listed in Table VII including both the upper and lower limits. One of the procedures for designing such a viscometer can be briefly described as follows: first specify the ranges of shearing stresses to be used which in turn determine the ranges of rates of shear, provided that the viscosity of the solvent is known. For example, the viscosity of water is 1.005 centipoise at 20°C or 0.894 centipoise at 25°C. Thus the magnitude of G is roughly a hundred times that of T for pure water. Next estimate the desirable flow time, t, for each bulb and also its volume, V , say, between 1 to 5 ml. These in turn determine the radius, R, of the capillary according to Eq. (44b). Thirdly adjust the ratio of A P / L or A h / L so as to obtain the desired T according to Eq. (44a). With the radius fixed both T and G remain unchanged so long as the ratio A h / L and V / t are kept constant respectively. Thus for viscometer I1 in Table VII the length of the capillary and the pressure head can be reduced to, say, 80 cm and 5 cm (for the upper bulb) without affecting the value of T . Likewise the
386
JEN TSI YANG
flow time can be either increased or decreased by proportional increase or reduction of the volume of the bulb. [It is noted however that the kinetic energy correction remains the same in both cases since it is only related to the volume flow rate according to Eq. (55b) .] Viscometers I1 and 111in the table merely illustrate the different ranges of shearing stresses that can be reached. As a rule a smaller radius and shorter length of the capillary are used for high-gradient viscometers. With some simple calculations one can easily design a capillary viscometer which serves the particular purpose of any experimental work. The dimenTABLEV I I The Dimensions of Three Hypothetical Multibulb Ilbbelohde Viscometers Dimension Length, L (cm) Radius, R (cm) Pressure head, Ah (em) (1) Upper bulb (2) Lower bulb Volume of flow, V (cm3) (1) Upper bulb (2) Lower bulb Flow time, t (sec). (1) Upper bulb (2) Lower bulb Shearing stress, T (dynes cm-2) (1) Upper bulb (2) Lower bulb Rate of shear, G (sec-')a (1) Upper bulb (2) Lower bulb a
I
I1
400 0.061
400 0.032
20 4 5 1 170 170 1.5 0.3 167 33
25 5 1 0.2 350 350 1 0.2 110 22
I11
4 0.016 25 5 5 1 280 280 50 10 5,500 1,100
Based on water at 25°C.
sions of the precision-bore tubing and bulbs can be calibrated with pure mercury. The viscometer should further be tested with water or other liquids of known viscosities and the corresponding shearing stresses and rates of shear should compare favorably with those calculated from mercury calibration. This test is especially important when a very long capillary is used, which by necessity is very likely bent into loops or other compact shapes. By so doing the cross section of the capillary might be subjected to distortion and a mean radius should be determined and used in Eqs. (44a and b). A slightly different procedure is adopted for the design of viscometer I in which a rather unusually large radius of the capillary is first specified. The flow time for each bulb can be kept fairly high by using a large volume
VISCOSITY OF MACROMOLECULES
387
of the bulb and also by lowering the shearing stress and rate of shear. Such a viscometer is desirable when the danger of clogging the capillary with solid particles (through surface denaturation, etc. ) becomes a problem.
E. Viscosities of Extremely Dilute Solutions Streeter and Boyer in 1954 reported that the reduced viscosity, qsp/C, of polystyrene exhibited a marked upward curvature in the Huggins’ plot when the concentration diminished below a certain critical concentration but it passed through a maximum and dropped again at still lower concentrations. Two years earlier Takeda and Tsuruta (1952) had found a similar behavior for the viscosity of polyvinyl chloride, although their paper was unnoticed at that time. These reports have rekindled an old controversy on the concept of a so-called “critical concentration” (Staudinger, 1932). According to one school of thought the properties of polymer solutions should undergo a marked change at this critical concentration; above it the polymer chains cannot move independently of one another whereas below it this molecular interference disappears. As a consequence many physical measurements in the range of concentrations commonly employed have been suspected to be of doubtful value. The upturn in the reduced viscosity below the critical concentration can be attributed partly t)o the untangling and partly to the expansion of the polymer chains, whereas its downturn at still lower concentrations can be due to the loss of polymer molecules by adsorption on the capillary wall of the viscometer. A second school, however, argues that various interactions have already manifested themselves in the concentration-dependence expressions of these physical properties. The common practice of extrapolation to zero concentration of a power series, for example, in the Huggins plot, should remain valid both above and below the critical concentration. Thus there is no need for a drastic revision of the whole concept of dimensions of polymers in solutions as implied by the former school. The “abnormal” viscosity behavior of the polymers at extremely low concentrations could very well be explained by adsorption alone. Numerous experimental results support one school over the other. As is often true for a controversial subject many of the data quoted as evidence were so conflicting that they caused further confusion of an already confusing topic. True, the experimental difficulties are extremely great, since the very nature of extremely dilute solutions demands special instrumentation of highest precision. In the case of viscosity measurements, problems of temperature control, atmospheric pressure fluctuation, cleaning, etc. become far more serious than in routine work. For example if qsp/C = 1 at C = 0.001 % the flow time must be reproducible to the order of one part in 100,000 for the desired accuracy. At present there is still no agreement as to whether adsorption is the
388
JEN TSI YANG
only cause for the “abnormal” behavior of the viscosity as described above (see, for example, Umstatter, 1954, 1958; o h m , 1955, 1958; Claesson, 1960). The situation is indeed very complicated: adsorption leads to a loss in polymer concentration with a corresponding decrease in flow time, while the adsorption film on the wall narrows the radius of the capillary, which according to Eq. (44c) varies inversely with the fourth power of the flow time. At moderate concentrations the increase in flow time due to smaller radius, however, more than offsets the decrease from the first effect, which i n turn results in a sharper increase in the tls,/C. Assuming that the thickness of the film is r , then we have v,,I(obsd.)
=
qx(corr.) (1
+ 4 r/R)
(64)
where R is the radius of the capillary. The corrected qr,l can be obtained by measuring the viscosities in a series of viscometers of different radii and extrapolating them to I/r = 0 ( o h m , 1955; Takeda and Endo, 1956). There is much argument about the correct equation for adsorption and the actual picture of the adsorbed film. For example, it has been asserted that the amount of solute in an extremely dilute solution is insufficient for the formation of a thick, uniform adsorption film. Nevertheless one ran define an effective thickness of the adsorbed film, r, for Eq. (64). This adsorption also depends on the concentration employed in a manner compatible with the Larigmuir isot,herm. These discussions merely point, out the complications tlhat may arise from the viscosity measurements of extremely dilute solutions. So far as the author is aware no “abnormal” viscosity behavior seems to have been reported for proteins in extremely dilute solutions (((1 5%). Nevertheless it seems desirable t,o keep in mind this controversy and its possible complications in the interpretation of viscosity data.
VI. CONCLUSIONS In this review we have briefly discussed the theoretical and expcrimerital tLspectnsof both Newtonian and non-Newtonian viscosities of polymer solutions. To protein chemists one of the interesting developments is no doubt the re-examination of the (Newt,onian) viscosity treatments of protein solutions. There are many assumptions involved in the effective use of intrinsic viscosity measurements for evaluating the asymmetry of the protein molecules, however attractive the conventional treatment may have appeared for the past two decades. Carefully interpreted, the intrinsic viscosity (at zero gradient) can still provide a reasonable estimate of the axial ratios of the protein molecules. The concept of equivalent hydrodynamic volume, sound in principle, has put the viscometry of protein solutions in a proper perspective, although the quantitative aspects of this new approach still
VISCOSITY OF MACROMOLECULES
389
await extensive tests and the insensitivity of the proposed p- and &functions to variations in the axial ratio of the equivalent ellipsoid makes this task formidable. Viscometry has been one of the most powerful tools for detecting the conformational changes or “denaturation” of proteins, but intimate details of the process cannot be deduced from the viscosity alone. The use of a combination of hydrodynamic measurements such as those involved in the evaluation of the @-functionmay enable us to deduce whether such changes are primarily due to the changes in shape or in effective volume of the protein molecules, even though the inferences are only of a semiquantitative nature. Of greater importance is the coordination of viscometry with other physicochemical methods such as the optical rotatory power and deuterium-hydrogen exchange ; together, they can shed a deeper insight on the internal structures of proteins. The theories of non-Newtonian viscosity for rigid particles are all in good agreement and are satisfactorily established by experimental data. This recent development offers a new measure of the rotary diffusion coefficient and also the degree of polydispersity of the macromolecules. Like flow birefringence, however, the technique is useful only for highly asymmetrical particles which show significant gradient dependence of the viscosities. For the Newtonian viscosities of flexible chain polymers various theories and experiments appear to converge to a good agreement, at least in the limiting case of ideal solutions. Little, however, has been exploited on the effect of branching on the viscosities of polymer solutions and it is not known whether the present forms of these theories can be applicable to solutions of certain proteins having a high degree of flexibility. For the non-Newtonian viscosities of polymer chains present theories are in a stage of rapid progress. Experimentally the gradient dependence of the viscosities is found to be closely related to the expansion of the polymer chains. Moreover, the striking difference in the non-Newtonian behavior between rigid particles and flexible chains appears to furnish a new means for studying the conformational changes of proteins in solutioiis.
ACKNOWLEDGMENTS The major part of this review was written at the Centre de Recherches sur les MacromolBcules, Strasbourg, France. The author is indebted t o Professors C. Sadron and H. Benoit and their colleagues for their valuable assistance and warm hospitality during his stay there. It is a pleasure to thank Professor J. T. Edsall for helpful criticism and numerous suggestions of the manuscript. Thanks are also due the management of the American Viscose Corporation for providing the author the opportunity to complete his work on viscosities (1956-1959). The financial support from a John Simon Guggenheim Memorial Foundation Fellowship, the National Science Foundation (G-10471), and also in part the United States Puhlic Health Service (SF-435, A-4008) is gratefully acknowledged.
390
JEN TSI YANG
VII. APPENDIX(Tables VIII-XIII) TABLE VIII Viscosity Increments, v , as a Function of the Axial Ratios of the Ellipsoids of Revolution", Axial ratio
Prolate
Oblate
Axial ratio
Prolate
Oblate
1 1.5 2 3 4 5 6
2.50 2.63 2.91 3.68 4.66 5.81 7.10 10.10 13.63 17.76 24.8 38.6 55.2 74.5 120.8
2.50 2.62 2.85 3.43 4.06 4.71 5.36 6.70 8.04 9.39 11.42 14.80 18.19 21.6 28.3
50 60 80 100 125 150 200 300 400 500 600 700 800 900 lo00
176.5 242.0 400.0 593 .o 882.0 1222 2051 4278 7247 10,921 15,216 20,250 25,880 32,200 39,117
35.0 41.7
8 10 12 15 20 25 30 40 -~ ~
0
MehI et al. (1940). Sadron (1953b).
55.1
68.6 102.3 136.2 204.1
391
VISCOSITY OF MACROMOLECULES
TABLEIX Frictional Ratios, f/fo and , as a Function of the Axial Ratios of the Ellipsoids of Revolutiona*
r/ro
flfo
Axial ratio Prolate
Zko Oblate
Prolate ~
1 1.2 1.4 1.6 1.8 2 3 4 5 6 7 8 9 10 12 14 15 16 20 25 30 35 40 50 60 70 80 90 100 200 300
1 1.003 1.010 1.020 1.031 1.044 1.112 1.182 1.255 1.314 1.375 1.433 1.490 1.543 1.645 1.739
-
1.829 1.996 2.183 2.356 2.518 2.668 2.946 3.201 3.438 3.658 3.867 4.067
-
1 1.003 1.010 1.019 1.030 1.042 1.105 1.165 1.224 1.277 1.326 1.374 1.416 1.458 1.534 1.604 1.667 1.782 1.908 2.020 2.119 2.212 2.375 2.518 2.648 2.765 2.873 2.974 -
-
Oblate ~
1 1.505 2.340 3.395 4.638 6.061 9.401
1 -
-
25.86
1.132 1.464 1.843 2.240 2.645 3.471 4.305 5.143 6.407
41.80 61.05 83.45
8.519 10.64 12.75
-
13.37 17.94
-
-
-
137.3 202.9 279.9 465.9 694.5 2428 5085
Svedberg and Pedersen (1940), Cohn and Edsall (1943). Scheraga and Mandelkern (1953).
-
-
16.99 21.24 25.48 33.96
-
42.44 84.86 127.3
392
JEN TSI YANG
TABLEX 6 - and &Functions for Ellipsoids of Revolution" B Axial ratio
1 2 3 4 5 6 8 10 12 15 20 25 30 40 50 60 80 100
200 300 0
x
6
10"
Prolate
Oblate
Prolate
Oblate
2.12 2.13 2.16 2.20 2.23 2.28 2.35 2.41 2.47 2.54 2.64 2.72 2.78 2.89 2.97 3.04 3.14 3.22 3.48 3.60
2.12 2.12 2.13 2.13 2.13 2.14 2.14 2.14 2.14 2.14 2.15 2.15 2.15 2.15 2.15 2.15 2.15 2.15 2.15 2.15
2.50 1.93 1.57 1.37 1.25 1.17 1.07 1.02 0.990 0.959 0.923 0.904 0.893 0.880 0.870 0.865 0.859 0.854 0.845 0.841
2.50 2.42 2.34 2.20 2.10 2.03 1.93 1.87 1.83 1.78 1.74 1.71 1.69 1.67 1.65 1.64 1.62 1.62 1.60 1.60
Scherttga and Mttndelkern (1953).
393
VISCOSITY OF MACROMOLECULES
TABLE XI Shape Factors for the Calculations of Lengths of Prolate Ellipsoids Axial ratio
(PI 1 1.2 1.4 1.5 1.6 1.8 2 3 4
5 6 7 8 9 10 12 14 15 16 20 25 30 35 40 50
60 70 80 90 100 150 200 300
Viscosity‘ (P/v)l/a
0.7368 -
-
0.9493 -
1.112 1.347 1.509 1.626 1.718
-
1.850 1.943 2.009
-
2.086 2.180 2.245 2.295 2.366 2.420 2.459 2.520
-
2.564 2,641 2.692 2.761
Rotary diffusion coefficientb (JP)’‘3
1.Ooo
-
1.385 1.567 1.676 1.753 1.811
-
1.895
-
1.956 2.002
-
Translational diffusion coefficientc (FPz/3)
1.Ooo 1.126 1.239 1.341 1.435 1.521 1.871 2.132 2.330 2.513 2.661 2.791 2.904 3.008 3.186 3.340
2.057
-
-
3.472 3.691 3.917 4.098 4.249 4.384 4.607 4.788 4.940 5.076 5.193 5.297
2.123 2.171 2.209 2.267 2.292 2.343 2.395
-
2.433
-
2.544 2.606
-
L(=2a) = [ (600[q]M/Nr)(pz/v))l/*= 6.82 X 10-S([q]M)1’3(p*/v)1/3. L(=2a) = [(k/r)(T/qoe)(~p2)ll’3= 3.53 X 1 0 - ~ ( T / ~ o e ) 1 ~ 3 ( J p z ) 1 ~ 3 . L(=2a) = [M(1 - ~ p ) / 3 ~ ~ ~ N s l ( F = p1.76 ~ / ~X)10-2s[M(1- ~ p ) / q ~ s ] ( F por ~ ’= ~) (k/3r) (T/qoD)(Fpz/3) = 1.46 x 10-17(T/qoD)(Fp2//8).
394
JEN TSI YANG
TABLE XI1 Viscosity Increments, Y, as a Function of a for Various Axial Ratios of the Ellipsoids of Revolutions Prolate
Oblate
a
0.00 0.25 0.50 0.75 1.oo 1.25 1.50 1.75 2.00 2.25 2.50 3.00 3.50 4.00 4.50 5.00 6.00 7.00 8.00 9.00 10.00 12.50 15.00 17.50 20.00 22.50 25.00 30.00 35.00 40.00 45.00 50.00 60.00 a
16
25
27.18 27.15 27.07 26.94 26.76 26.54 26.28 25.98 25.66 25.32 24.97 24,24 23.49 22.77 22.07 21.41 20.22 19.19 18.30 17.53 16.86 15.51 14.42 13.69 13.05 12.52 12.06 11.32 10.74 10.27 9 A47 9.477 8.845
55.19 55.13 54.96 54.68 54.29 53.81 53.25 52.62 51.93 51.20 50.44 48.86 47.26 45.70 44,21 42.80 40.24 38.04 36.14 34.50 33.07 30.21 27.89 26.34 24.98 23.86 22.90 21.35 20.12 19.12 18.24 17.46 16.12
Scheraga (1955).
50
176.8 176.6 176.0 175.1 173.8 172.1 170.3 168.1 165.8 163.4 160.8 155.5 150.1 144.9 139.9 135.2 126.6 119.2 112.9 107.5 102.7 93.19 85.54 80.43 75.93 72.28 69.12 64.05 60.39 56.80 53.94 51.40 47.04
100
593.7 593.0 591 .O 587.7 583.2 577.6 571.1 563.8 555.8 547.4 538.5 520.3 501.8 483.8 466.6 450.3 421 .O 395.7 374.0 355.3 339.1 306.7 280.7 263.3 248.0 235.7 225.0 207.9 194.5 183.6 174.0 165.5 151.O
25
18.19 18.18 18.15 18.09 18.01 17.91 17.80 17.67 17.52 17.37 17.22 16.89 16.55 16.22 15.90 15.60 15.04 14.54 14.11 13.72 13.38 12.66 12.07 11.64 11.26 10.93 10,64 10.15 9.753 9.411 9.114 8.844 8.391
100 69.10 69.06 68.91 68.68 68.36 67.97 67.51 66.99 66.42 65.82 65.18 63.87 62.53 61.20 59.92 58.70 56.46 54.48 52.73 51.19 49.82 46.96 44.57 42.86 41.33 40.03 38.86 36.91 35.32 33.94 32.75 31.66 29.82
395
VISCOSITY OF MACROMOLECULES
hl./[vl.-o
TABLE XI11 as a Function of a for Various Axial Ratios of the Ellipsoids of Revolutiona Prolate
a
0.00 0.25 0.50 0.75 1.OO 1.25 1.50 1.75 2.00 2.25 2.50 3.00 3.50 4.00 4.50 5.00 6.00 7.00 8.00 9.00 10.00 12.50 15.00 17.50 20.00 22.50 25.00 30.00 35.00 40.00 45.00 50.00 60.00
Oblate
16
25
50
100
25
100
1.oooo 0.9989 0.9960 0.9912 0.9846 0.9765 0.9669 0.9559 0.9441 0.9316 0.9187 0.8918 0.8642 0.8378 0.8120 0.7877 0.7439 0.7060 0.6733 0.6450 0.6203 0.5706 0.5305 0.5037 0.4801 0.4606 0.4437 0.4165 0.3951 0.3779 0.3623 0.3487 0.3254
1.0000 0.9989 0.9958 0.9908 0.9837 0.9750 0.9649 0.9534 0.9409 0.9277 0.9139 0.8853 0.8563 0.8281 0.8011 0.7755 0.7291 0.6893 0.6548 0.6251 0.5992 0.5474 0.5053 0.4773 0.4526 0.4323 0.4149 0.3868 0.3646 0.3464 0.3305 0.3164 0.2921
1.oooO 0.9989 0.9955 0.9904 0.9830 0.9734 0.9632 0.9508 0.9378 0.9242 0.9095 0.8795 0.8490 0.8196 0.7913 0,7647 0.7161 0.6742 0.6386 0.6080 0.5809 0.5271 0.4838 0.4549 0.4295 0.4088 0.3910 0.3623 0.3416 0.3213 0.3051 0.2907 0.2661
1.oooO 0.9988 0.9955 0.9899 0.9823 0.9729 0.9619 0.9496 0.9362 0.9220 0.9070 0.8764 0.8452 0.8149 0.7859 0.7585 0.7091 0.6666 0.6300 0.5985 0.5712 0.5166 0.4728 0.4435 0.4177 0.3970 0.3790 0.3502 0.3276 0.3093 0.2931 0.2788 0.2543
1.0000 0.9995 0.9978 0.9945 0.9901 0.9846 0.9786 0.9714 0.9632 0.9549 0.9467 0.9285 0.9098 0.8917 0.8741 0.8576 0.8268 0.7993 0.7757 0.7544 0.7356 0.6960 0.6636 0.6399 0.6190 0.6009 0.5849 0.5580 0.5362 0.5174 0.5010 0.4862 0.4613
0.9994 0.9973 0.9939 0.9893 0.9837 0.9770 0.9695 0.9612 0.9525 0.9433 0.9243 0.9049 0.8857 0.8672 0.8495 0.8171 0.7884 0.7631 0.7408 0.7210 0.6796 0.6450 0.6203 0.5981 0.5793 0.5624 0.5342 0.5111 0.4912 0.4740 0.4582 0.4316
1.oooO
Calculated from Scheraga (1955). See also Yang (1958a). For axial ratios other than those listed here see t h e original papers.
396
JEN TSI YANG
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OPTICAL ROTATION AND THE CONFORMATION OF POLYPEPTIDES AND PROTEINS' By PETER URNES and PAUL D O T Y Department of Chemistry, Harvard University, Cambridge, Massachusetts
I. Introduction., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 11. The Analysis of Rotatory Dispersion Curves., . . . . . . . . . . . . . . . . . . . . . . . A. Expressions for Optical Rotatory Power,. . . . . . . . . . . . . . . . . . . . . . . . . B. Origins and Use of the Drude Equation., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 C. The Moffitt Equation for the Optical Rotation of Helical Structures. . . . 413 D. Optical Rotation in Regions of Absorption: The Cotton Effect. . . . . . . . . . 419 . . . . . . . . 424 111. The Optical Rotatory Dispersion of Synthetic Polyp A. The Establishment of Chain Conformation in Pol . . . . . . . . . . . . 425 B. The Moffitt Parameters for Helical Polypeptides, C. The Rotatory Contribution of the Helix., . . . . . . . 1. The Comparison of Helical Dispersion with That of a Disordered Chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Meso-Helices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 D. The Issue of Helical Sense.. . . . . . . . . . . . . . . . . E. Optical Rotatory Dispersion of Polypeptide F. Synthetic Polypeptides with Unusual Rotat G. The Moffitt Equation for Mixtures of Helices ............. H. Helix-Coil Transitions,... I. Rotatory Properties of th IV. The Optical Rotatory Dispersion of Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 481 A. The Compact, Rigid Structure of Globular Proteins.. . . . . . . . . . . . . . . . . . 482 B. Evidence for the Existence of Helices in Globular Proteins.. . . . . . . . . . . 483 C. Points of Similarity between Protein and Polypeptide Dispersions.. . . . 485 1. Denatured Proteins and Random Coils.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 490 491 2. Fibrous Proteins and Helical Polypeptides. . . . . . . . . . . . . . . . . . . . . . . . . 3. Helix-Coil Transitions in Nonaqueous Solvents. . . . . . . . . . . . . . . . . . 494 4. Changes in the Specific Rotation upon Denaturation.. . . . . . . . . . . . . . 49G 5. Degree of Folding in Globular Proteins and the Rotatory Dispersion of Helices.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,499 D. Scales of Helical Content as Applied to Proteins.. . . . . . . . . . . . . 504 E. Helical Content from Rotatory Dispersion and Other Methods. . . . . . . . . . 509 F. Disulfide Bonds and Prosthetic Groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513 G. Rotatory Deviations from the Helix-Coil Pattern. . . . . . . . . . . . . . . . . . . . . 516 H. Optical Rotation and Studies of Tertiary Structure. . . . . . . . . . . . . . . . . . . 525 1 The preparation of this manuscript and a number of the investigations reported in it were aided by the National Science Foundation (G-7487, G17556) and the Office of Naval Research (N5 ori-07654).
40 1
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I. Collagen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Cotton Effects.. . . . . . . . . . . . . . . . . . . .............................
528 529 V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534 References. . . . . . . . . . . . . . . . . . . . . ........ . . . . . . . . 536
I. INTRODUCTION Optical rotatjion and chain conformation in polypeptides and prot>eiiis have their common ground in the peptide bond, for it is the spatial disposition of t,his generic chemical group that gives rise to the principal rotatory characteristics of these substances and at the same time defines their secondary structure. Changes in the mutual orientation of peptide groups in the polymeric backbone are thus tantamount to conformational changes, so that the special sensitivity of optical rotatory power to steric form renders it, uniquely suited to the study of conformation. It is common practice to use rotatory power a t one wavelength to detect structural alteration, as in the study of helix-coil transitions and denaturation, but monochromatic data often do not alone permit a distinct specification of the change that takes place. Optical rotatory dispersion, that is, the variation of optical rotatory power with wavelength, can in principle provide a greater amount of information that may be correlated with structure and therefore offersa basis for more perceptive studies of conformation and conformational change. A natural consequence of the discovery of helical struct,ures in synthetic po1ypept)idesand proteins was the prominence that helices acquired in both theoretical analysis and experimental investigation of the rotatory dispersion of these substances. One of the early successes of this approach was the demonstration that, the helical conformation of a polypeptide chain endows the optical rotatory dispersion with qualitatively different features than it otherwise possesses. This in turn led to the attempt to interpret the dispersive properties of native proteins as a result of a simple partitioning of tjhe peptJide groups into helical and disordered regions. This is an attractive hypothesis because it provides a general plan for the compact, folded form that globular proteins exhibit in solution, a form that is intimately related to their biological function. Furthermore, it has received clear support from t,he recent crystallographic investigations of Kendrew and his collaborators that have provided its first direct test (Kendrew et al., 1960, 1961). This work shows that crystalline myoglobin contains 77 5% of its residues in a number of relatively short a-helices connected by segments devoid of obvious order. Although a large number of globular proteins permit a consistent analysis of their rotatory behavior in terms of disordered regions and helices of a single sense, the simplicity of this hypothesis has invited exceptions. With the growing number of rotatory dispersion studies of proteins, several cases
OPTICAL ROTATION AND PROTEIN CONFORMATION
403
have emerged in which it appears insufficient to explain the observations. This suggests the occasional occurrence of conformations other than those envisaged in the hypothesis. Of the possible candidates, two hydrogenbonded structures, helices of opposite sense and the extended or p-form, seem likely. These two forms have now been partially characterized by optical rotation, and the extent to which either or both of them can account for deviations from the simple model has become a subject of current investigation. Since the dispersive properties of helices in what will be taken as the standard sense, now known to be right-handed as in myoglobin, have been the most thoroughly studied and since a case can be made that it is the predominant sense in proteins, this review will focus on the capacity of optical rotatory methods to discern mixtures of this conformation with disordered regions. It will discuss the manner in which theoretical considerations have provided the forms into which rotatory data are currently cast, the calibration of their constants by studies of synthetic polypeptides in known conformation, and then the application of these equations and scales in the structural interpretation of the rotatory dispersion of proteins. This pattern of analysis will undoubtedly undergo refinement and revision as these methods are applied to new species of polypeptide and protein in concert with other means of conformational assignment. In particular, an extension of the spectral range of measurement toward optically active absorption bands in the far ultraviolet can be expected to yield new information about the rotatory power of the peptide bond and thus enhance the interaction of theory and observation that has already proved fruitful. Recent reviews of the optical rotation of proteins have been made by Jirgensons (1961a, d), Leach (1959), ShootJer (1960), and Schellman and Schellman (1958, 1961), while those of Blout (1960), Katchalski and Steinberg (1961), Kauzmann (1957a), Todd (1960), and Yang (1961) include synthetic polypeptides as well. Early developments in the field are discussed by Doty and Geiduschek (1953). A review by Harrington and von Hippel devoted to collagen, including its rotatory properties and those of poly-L-proline, appears in this present volume. 11. THEANALYSISOF ROTATORY DISPERSIONCURVES Let us begin by examining the optical rotatory dispersions of two typical synthetic polypeptides and a well-studied protein, as illustrated in Fig. 1. The spectral range along the abscissa is that usually available for polarimetry, while the ordinate is the reduced mean residue rotation, a quantity that will be defined by Eqs. (1) and (4).For each of the three substances, two different conformations are represented. Those denoted by PBG refer to poly-y-benzyl-L-glutamate, which exists as a flexible, random coil in
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PETER URNES AND PAUL DOTY
dichloroacetic acid but! adopts a completely helical conformation in less polar solvents such as dioxane and m-cresol (Doty et a,?., 1956, Yang and Doty, 1957). Its ionic, water-soluble derivative, poly-L-glutamic acid, +
‘
0
°
2
:: ::
-400.
8 ?: I
I
I
I
I
Fro. 1 . Optical rotat,ory dispersions for ordered and disordered forms of two synt,hetic polypeptides and a globulttr protein. PBG : poly-ybenzyl-L-glutamate. Helical structure in m-cresol, .---a; random coil in dichloroacetic acid, 0-0. (Yang and Doty, 1957.) PGA: poly-L-glutamic acid. Helical structure at pH 4.72 in 1:2 water: dioxane mixture, 0.2 M NaCl, A--A; random coil a t pH 6.56 in the same solvent, A - A. (Doty et d.,1957.) B8A: bovine serum albumin. Native protein in water a t pH 5.47, .---H; denatured protein in 8 M urea a t pH 5.5, 0 - 0 . (Dispersion calculated from values of [alasp and xc measured by Schellman, 1958d.)
has conformational properties that are responsive to pH, for as the pH is lowered with progressive neutralization of the side chains, the helical conformation becomes stable (Doty et a,?.,1957). These two forms are indicated by PGA. The dispersion of bovine serum albumin, BSA, is shown in both its native and denatured states (Schellman, 1958d). It can be seen that the rotations of the helical forms are more positive than t,hose of the disordered
OPTICAL ROTATION AND PROTEIN CONFORMATION
405
chains, as are those of the native protein compared to its denatured form, and the shape of the helical dispersions shows distinctly more curvature than that for random coils. Yet, in contrast to absorption spectra with their correspondence to discrete chemical groups, these curves are relatively featureless, so that the way in which they may be treated to yield structural information becomes a matter of peculiar methodological concern. Two equations developed from theories of optical activity have played an essential role in the treatment of dispersion data of polypeptides and proteins. The Drude equation can be derived from general principles of optical rotatory power and hence purports to describe the rotatory dispersion of all substances in regions distant from optically active absorption bands, whereas the Moffitt equation has been formulated with specifically helical structures in mind. No attemptJ will be made to develop rigorously the theoretical background of these expressions. The emphasis here will rather be upon the form which they dictate for rotatory dispersion and the accommodation which their usage entails between the theoretical and empirical significance of their parameters. For discussions of the general theory of optical rotatory power, reference should be made to the review of Schellman (1958a) and the somewhat fuller treatments of Condon (1937) and of Heller and k'itts (1960) as well as the texts of Born (1933), Eyring et al. (1944), and Kauzmann (1Y57b).
A. Expressions for Optical Rotuloru Powcr As will be seen, the rotatory dispersions in Fig. 1 may he distinguished and yuantitat,ively compared through graphical treatment according to the Drude and Moffitt equations. In order for this type of comparison to be carried out, however, the optical rotatory power of these different substances a t individual wavelengths in the dispersion must be placed on a common basis. For this purpose, several expressions may be used. The specific rotation, [a]x , expresses the observed rotation, ax, in degrees a t wavelength A, in terms of the path length in decimeters, d, and the weight concentration of optically active solute, c', in grams per milliliter or thc more common measure c in grams per 100 ml. [alx =
1
-a x
dc'
=
~
100 ah dc
Since optical rotatory power is a function of both wavelength and temperature, it is necessary to specify these with any stated value. The molar rotation, [ M I A ,
in which M is the molecular weight of the solute, is more useful for corn-
406
PETER URNES AND PAUL DOTY
paring substances of different molecular weight, but since the optical rotatory power of polymeric substances is principally a function of individual residues rather than the total molecular weight, the mean residue weight provides the most satisfactory basis for comparing polymers that differ both in amino acid composition and molecular weight. The m a n residue rotation is therefore defined by
in which MRW is the mean residue weight. This quantity has the dimensions degrees centimeters2 per decimole. Most data for proteins exist as specific rotations and can be used for comparative purposes within this class of molecules because the mean residue weights cluster about 115. However, these values should be expressed as mean residue rotations to bring them into concordance with synthetic polypeptides when careful analyses are attempted. Optical rotatory power is also dependent upon the refractive index of the solvent, so that to reduce the observed rotations in a variety of solvents to a common environment, they are brought to what they would approximately be under vacuum conditions by the Lorentz correction factor. The inclusion of this correction leads to the reduced mean residue rotation,
in which n is the refractive index of the solvent a t wavelength A. It is this quantity that is generally employed as a comparative measure of the rotatory power of polypeptides. For the most precise work, the dispersion in refractive index of the solvent must also be taken into account in evaluating [m’]A . This is usually done with t,he aid of the Sellmeier approximation,
which requires measurement of the refractive index a t two wavelengths in order to calibrate the two constants a and A,. This correction can be significant in nonpolar and denaturing solvents.
B. Origins and Use of the Drude Equation The equation initially proposed by Drude (1900) to describe rotatory dispersion in spectral regions far from optically active absorption bands is [alx =
c
Ki
i
in which X i are the wavelengths of the optically active electronic transi-
OPTICAL ROTATION A N D PROTEIN CONFORMATION
407
tion, Ki are constants proportional to the rotatory strength of the transsitions, and X is the wavelength of incident radiation. Each term in the summation represents the contribution of a single transition, and this contribution is called a partial rotation. This equation was originally derived from a model consisting of an electron oscillating along a helical path. Although Born (1918) and Kuhn (1933) in their classic theoretical work point out that this model is actually incapable of generating optical activity, other treatments of rotatory dispersion lead to the same functional relationship. The following sketch will delineate the origins of the form of the Drude equation and the kind of information that is in principle obtainable from its application to rotatory dispersion data. It is possible to explain the rotation of a plane of polarized light in terms of different velocities for the right and left circularly polarized components into which it may be resolved, and hence ascribe optical activity to different indices of refraction of the material medium traversed (Condon, 1937; Moscowitz, 1960a). The ultimate aim, however, of a theory of optical activity is to refer it to the structure of molecules which comprise the material medium. A molecule is endowed with rotatory power by virtue of asymmetry, two varieties of which are relevant to proteins and polypeptides (Heller and Fitts, 1960). The most common asymmetry is that imposed by a carbon atom with four different substituents, each one of which exists in an unsymmetrical environment provided by the other three together with the central carbon atom. Yet a simpler type of asymmetry is that of identical groups disposed in a helical array, a type of order graphically depicted a century ago by Yasteur (1860). “Imagine a spiral staircase whose steps are cubes, or any other objects with superposible images. Destroy the stair and the dissymmetry will have vanished. The dissymmetry of the stair was simply the resuIt of the mode of arrangement of the component steps.” This type of arrangement is frequently designated by the term “conformation” in order to distinguish it from “configuration,” which is reserved to describe the D- and L-forms in which four different groups can attach to a carbon atom and which would thus characterize individual steps in Pasteur’s staircase. In a parallel distinction, the term “asymmetry” is sometimes confined to the handedness involving the valence angles of a central atom, while “dissymmetry” is used to indicate features of over-all molecular arrangement. The dissymmetry of a helix is hence expressed in two enantiomorphic conformations, right-handed and left-handed. Structural asymmetry and dissymmetry can be made amenable to theoretical treatment by showing in a general way that electromagnetic waves can perturb charged particles, of which molecules are constructed, so as to produce rotatory phenomena. A charged particle, which we may take to be a loosely bound electron for the reason that rotatory power originates almost entirely from electronic rather than nuclear motions, will oscillate
408
PETER URNES AND PAUL DOTY
in response to the changing electromagnetic field of the incident radiation, and since moving charges themselves generate electromagnetic fields, the radiation which they emit in the form of secondary wavelets can interact with the primary incident radiation. If a component of the secondary wavelets is polarized perpendicularly to the primary wave, its superposition will have the net effect of rotating the plane of polarization. The amount of rotation will hence be a function both of the frequency of the incident radiation and the natural frequency with which the particle oscillates, thus giving rise to rotatory dispersion. One way of picturing how the appropriate spatial and phase relationships might arise is to imagine that the electron is not free to move in the direction in which the primary wave is polarized but rather is constrained in an unsymmetrical path because it is subject to the unsymmetrical electromagnetic environment provided by other charges in the molecule. This motion of the electron can generate appropriate secondary wavelets to cause the rotation of the incident beam. The application of electromagnetic theory to this kind of interaction with random orientation in solution leads to the following expression for the sperific rotation of a substance of molecular weight M at wavelength A :
where N is Avogadro’s number, n the refractive index of the medium, and molecular parameter that characterizes the induced electric arid magnetic moments responsible for the secondary wavelets. /3 is a single term representing the entire complex of electronic effects within a molecule that endow it with optical activity and hence is a theoretical measure of structural asymmetry. Unless these effects can be calculated from the known electronic structure of a molecule in order to reach a value for @, no prediction of the magnitude or sign of the rotation can be made. Rosenfeld (1928) has related p to the electronic structure of any molecule by showing that it is a function of discrete electronic transitions and hence is related to the electronic absorption spectrum of the molecule. For regions far from the optically active absorption bands, the following equation may be derived from quantum mechanical perturbation theory,
@ is a
c
Ri
@=&-
in which v; is the frequency characterizing an electronic transition to an excited state, c is the velocity of light, h is Planck’s constant and v is the frequency of incident light. Ri is designated the rotatory strength of the transition and depends upon the electric and magnetic moments associated with it. Equation (8) may be substituted into Eq. ( 7 ) to give an expression for the specific rotation,
OPTICAL HOTATION A N D PROTEIN CONFORMATION
n2
=
+2
(9600mN)
( 7 MhX')
409
Ri
and, following the treatment of Schellman (1958a), the various constants can be condensed into a new variable, Ai , and the frequencies replaced by the corresponding wavelengths to give the expression
The form of the dispersion is hence seen to be identical with that in the original Drude equation, for it is characterized by the term (X' - A:)-', which is a feature shared by all resonant systems in forced oscillation (Joos, 1934). If we knew the rotatory strength and wavelength of every optically active transition in a particular molecule, we could then predict the dispersion of the specific rotation in regions of relative transparency. Conversely, this equation suggests that in principle one can estimate the electronic parameters of individual partial rotations from the observed dispersion. This kind of detailed correspondence between structure and rotatory dispersion has rarely been achieved, both because an adequate theoretical knowledge of the electronic structure of molecules is still lacking and, as will be seen in what follows, dispersion curves are, in general, sufficiently featureless to preclude the computation of more than a few constants. The observed dispersions of many substances in the visible spectrum obey the simple Drude equation,
which consists of a single term of Eq. (10) in which X i is designated A, and A : , X i and the refractive index term are condensed into the constant A . The criterion for the adequacy of this simple equation is a linear plot of the data when they are expressed in one of several alternative rearrangements of its form. The accuracy with which the constants A, and A may be obtained does, however, depend upon the graphical procedure employed. In the procedure of Lowry (1935), l/[aIxis plotted against A', the rotation constant A determined from the slope and the dispersion constant A, from the intercept and the value of A . This type of plot has the drawback that X, depends upon an intercept near zero which is difficult to specify with precision and, further, that ultraviolet data, which are more accurate than those in the visible spectrum because of inherently greater rotations, are concentrated into a small region, Heller ( 1958) recommends plotting 1/[a]AX2 against ]/A2, obtaining A from the intercept and X, from this value and the slope, thus enhancing the short wavelength measurements. The method proposed by Yang and Doty (1957), in which [a]hX' is plotted
410
PETER URNES AND PAUL DOTY
against [(Y]A ,shares this advantage and in addition permits A, to be obtained directly from the slope without independent calculation of A . In this instance, the measurement of slopes is more discriminating than that of intercepts, and further, the discontinuity in reciprocal plots of [a]A at [ol]A = 0 is avoided. For characterizing a given dispersion, A, is always computed, but the rotation at a reference wavelength, [ ( Y ] A , is in fact more often reported than A and, for purposes of describing the dispersion, serves as a parameter equivalent to it. If any two parameters of a simple Drude dispersion are specified, then the third can always be calculated. It must, however, be borne in mind that rotations at conventionally high reference wavelengths, 546 and 589 mN, may be somewhat discounted in obtaining the best linear fit, so that [(Y]A may not be as representative of the dispersion as the constant A . The existence of a unique value for A, does not imply that a single electronic transition causes the observed rotation, for the sum rule derivable from the theory of optical rotation,
CRi=O
(12)
shows that there must be at least two optically active transitions in any molecule that possesses rotatory power. For this reason, as Schellman (1958a) points out, the constants A, and A must represent statistical averages of a number of terms, and adherence to the simple Drude equation therefore cannot yield accurate information on individual partial rotations. A, is sometimes taken to indicate the position of the partial rotation which dominates the visible rotations, but this is true only in favorable circumstances in which a strong rotatory band exists at relatively long wavelengths, for example, that of the carbonyl group in certain ketones. In instances in which dispersion data have been compared with ultraviolet absorption spectra (Lowry and Hudson, 1933; Singh and Saxena, 1960), there is no correspondence between A, and absorption bands, though it must be acknowledged that not all absorption bands are optically active and that strong rotations may be associated with bands of very weak intensity (Kuhn, 1958). An effective rotatory strength, R, , associated with A, can be computed from A by means of Eqs. (9) and ( l o ) , but its significance is subject to the same qualifications as that of A,. All dispersion in regions distant from optically active absorption bands will in principle fit a single term equation for mathematical reasons that have been brought out by Condon (1937), Moffitt and Yang (1956), and by Schellman (1958a). Schellman shows that if A, is a wavelength in the vicinity of rotatory bands denoted by X i , then a series expansion of Eq. (10) about (A2 - At)-' will converge rapidly at high wavelengths of in-
OPTICAL ROTATION A N D PROTEIN CONFORMATION
41 1
cident light, A. If an appropriate definition of A, in terms of X i and Ai is made [see Eq. (22)], the second term vanishes altogether, leaving only higher order terms that are negligible and the first term. Thus the theoretically valid summation in Eq. (10) can be shown to approximate to the simple phenomenological form of Eq. (11) at sufficiently high wavelengths, so that the series expansion serves as a formal device to bridge theory and experiment. The fact that visible dispersions indeed obey the simple form +
20
I
I
I
I
I
I
I
I
, x" -201
a--
-60
is therefore to be expected and in no way detracts from the empirical support they give to the validity of the form of the Drude equation. As an illustration, the rotatory dispersions shown in Fig. 1 have been replotted in Fig. 2 in a manner that yields linear behavior when the simple Drude equation is valid. It is seen that the disordered forms of the three substances do yield straight lines conforming with Eq. (10). The native form of bovine serum albumin does likewise, but the two purely helical structures, in contrast, exhibit distinct curvature. The behavior displayed by the helical forms in Fig. 2 is formally denoted
4 12
PETER URNER AND PAUL DOTY
as complex dispersion (Lowry and Dickson, 1914). Such nonconformity with the simple Drude equation has been observed in other systems, especially as measurements are extended toward optically active absorption bands. One would expect the one-term equation to fail as the rotatory bands are approached, hut, it is worth noting that, the wavelength region in which curvature becomes apparent is a relative matter that depends upon the graphical procedure. For example, with the method used in Fig. 2 for the dispersion of helical poly-L-glutamic acid, deviations from the best straight line appear at high wavelength, whereas the same data display curvature at low wavelength when treated by the method of Lowry, which spreads out the high wavelength points. Complex dispersion can generally be made amenable to quantitative assessment by adding a second term to the simple Drude equation, since the two additional constants thereby provided are adequate to express the data in linear form. Thus, for empirical comparisons, equations such as
and an abbreviated form,
are found useful, and the graphical and algebraic techniques employed have been discussed by Lowry (1935) and Heller (1958). The constants derivable from these expressions are not necessarily more representative of the actual rotatory bands than are those from a single term plot (Singh and Saxena, 1960), so that they must be interpreted with like caution. The cases of complex dispersion shown in Fig. 2 can be handled in this way (Yang and Doty, 1957), but a more systematic approach became possible at the same time as complex dispersion was first observed for helical polypeptides (Doty and Yang, 1956). This is the treatment] originating in the theoretical work of Moffitt (1956a). Before proceeding to this analysis, two generalizations, together with the fundamental questions to which they lead, must be emphasized. The first is that most helical polypeptides exhibit complex dispersion, while as random coils their dispersion is simple. The complexity is a distinct consequence of the formation of the helix rather t,han of solvent changes or ionization. How, therefore, does the helical structure give rise to complex dispersion? And, if in polypeptide systems complex dispersion is a phenomenon with features peculiar to helices, can these features be employed as unequivocal signs for the presence of helices? The second general point is that globular proteins display simple dispersion in both the native and
OPTICAL ROTATION AND PROTEIN CONFORMATION
413
denatured states over the same spectral range in which the dispersion of helical polypeptides is obviously complex. Values of [m’]589and X, for most protein dispersions both decrease on denaturation and tend to reach values shared by synthetic polypeptides in the random coil conformation. In what way, then, may the simple dispersive properties of native proteins be analyzed to determine whether these molecules contain ordered structures known to exist in their synthetic analogues? The development of the Moffitt equation for the complex dispersion of helices and its experimental adaptation to protein dispersions has provided a basis for answering these questions, so that it is to this expression that we now turn.
C . The Mojitt Equation for the Optical Rotation of Helical Structures I n 1956 theoretical interest in the relation of molecular structure to rotatory dispersion was awakened by Moffitt’s analysis of the rotatory dispersion of helices (Moffitt, 1956a). Although it later required revision, this theory was of germinal importance in producing quite useful predictions about the dispersive properties of helical macromolecules. The a-helix of polypeptide systems is defined by the periodic disposition of identical bonds in a rigid, helical array that may be regarded as a molecular crystal. Moffitt’s general treatment showed that identical chromophores in a helical arrangement may interact to form an exciton system such that any one transition of an individual chromophore splits into two transitions that are Characteristic of the helix as a whole, one of which is parallel to the screw axis and the other perpendicular. This splitting leads to two major contributions which are of equal and opposite rotatory strength but which do not cancel because they occur a t different wavelengths. The partial rotation per residue contributed by this pair in regions far from the absorption band concerned may be expressed in a two-term Drude equation,
in which XIIi and XLi are the wavelengths of the respective transitions. This expression may be recast by suitable approximation and definition of constants into a form in which the individual wavelengths are represented by their average, X i , and the difference between them incorporated into the constant bi . This latter constant then becomes a measure of the splitting and the chromophoric interaction characteristic of the helix.
This form will give complex dispersion, but since the same dispersion constant, X i , occurs in botjh terms, it is the unusual wavelength dependence
414
PETER URNES AND PAUL DOTY
of the second term, which contains the inverse square of (A2 - A?), that gives rise to the complexity. The sum of the partial rotations is the total rotation per monomer unit,
This expression is analogous to the general Drude formula (lo), but it suggests that, in a spectral region distant from the optically active transitions of a helical polymer, the dispersion will be complex rather than simple. In order to apply this general relationship to the empirical data of helical polymers, Moffitt and Yang (1956) then developed a phenomenological equation in which the above sums are replaced by single terms and thus derived an expression that is analogous to the simple Drude equation.
Much as Eq. (10) can be expanded in inverse powers of (Az -A:) to yield the simple form, Eq. (17) can be expanded in inverse powers of (Az - Xi), the constants a. , bo , and A0 defined in terms of a; , bi , and X i , and higher order terms neglected. In the light of the theory and the approximations from which this equation is developed, the constants bo and Xo are principally, though not entirely, functions of the helical skeleton alone and accordingly should be relatively insensitive to environmental factors such as solvent changes and the nature of side chains. The constant a0 , on the other hand, represents both the intrinsic residue rotations, which would be present irrespective of the helix, and interactions within the helix. Since intrinsic residue rotations are known to vary with environment, a0 should likewise be expected to vary. Moffitt’s prediction that the dispersion of helical polypeptides in the visible spectrum would be complex instead of simple found immediate support in the complex dispersion of helical poly-ybenzyl-L-glutamate observed by Doty and Yang (1956). Yet the issue at stake in the first application of Eq. (18) by Moffitt and Yang (1956) was not whether it would adequately describe complex dispersion, for Xo was chosen as that wavelength which would give a linear obedience of the data when [m’]x(X2- A:) was plotted against (A2 - Xi)-1. The attempt was rather to determine if the constants a. and bo , which are derived from the intercept and slope, respectively, of this plot, and Xo behaved as the theory stipulated. It was found, in agreement with theory, that bo and Xo for poly-7-benzyl-L-glutamate and poly-L-glutamic acid in the helical conformation were in fact relatively insensitive to solvent, temperature and, insofar as these polymers differ, to side chains, while a0 did vary with these conditions. Furthermore,
OPTICAL ROTATION A N D PROTEIN CONFORMATION
415
in the random coil conformation these polymers exhibited simple dispersion so that the second term in Eq. (18) and its coefficient bo were not required to express the data in linear form, as might be expected for the absence of helical structures. The quantitative aspects of this theoretical approach fared less well, however, in Moffitt’s attempt to determine bo and XO (Moffitt, 1956b). On the basis of the far ultraviolet spectra of simple amides and peptides (Hunt and Simpson, 1953; Ham and Platt, 1952; Peterson and Simpson, 1955), he selected two absorption bands at 148 and 185 mp as the electronic transitions most likely to contribute strongly to rotatory dispersion in the visible and near ultraviolet spectrum. Given reasonable estimates of the oscillator strengths and polarizations of these transitions, the geometry of the a-helix as proposed by Pauling et al. (1951) and the assumption that it is right-handed, Moffitt computed the splitting produced by chromophoric interaction within each of these band systems and hence could calculate bi characteristic of each X i . Combination of the various constants for both bands led to Xo = 200 mp and bo = -580. A left-handed helix would give bo of the same magnitude but of opposite sign. The XO determined experimentally by Moffitt and Yang (1956) is 212 f 5 mp, and bo values cluster about -630, not only for the two helical structures initially studied but far a wide variety of helical polypeptides that will be discussed in greater detail below. This close agreement cannot, unfortunately, be taken as support for the proposition that rotatory dispersion in the visible spectrum is dominated by these two bands or that these helices are righthanded, for the joint critique of Moffitt et al. (1957) points to the omission of critical interactions in Moffitt’s treatment that vitiates quantitative prediction. Another line of theoretical endeavor aimed at calculating the rotatory parameters of the a-helix has, like Moffitt’s work, proceeded from the coupled oscillator theory of optical activity developed by Born (1915) and by Kuhn (Kuhn, 1929; Kuhn and Freudenberg, 1932), but it makes explicit application of Kirkwood’s polarizability theory of rotatory power (Kirkwood, 1937; Schellman, 1958a) instead of an exciton treatment. Fitts and Kirkwood (1956a) first computed the specific rotation for a right-handed a-helix of polyglycine and on this basis found reasonable agreement with the change in specific rotation observed in helix-coil transitions of a number of polymers (Fitts and Kirkwood, 1956b), a change which was assumed to reflect only the destruction of the helix but which has since been shown to depend as well upon solvent effects (Yang and Doty, 1957). By a subsequent calculation of the visible dispersion based upon Moffitt’s choice of active chromophores (Fitts and Kirkwood, 1957), they obtained excellent correspondence with optical rotations determined by Doty and Lundberg
416
PETER URNES AND PAUL DOTY
(1 957) for the meso-helix of poly-y-benzyl-L-glutamate (see Section 111, C, 2 ) , data which are presumably free of solvent effects in that no helix-coil transition is involved, and found that the calculated dispersion is complex. 96" is remarkably close to Although their computed value of [172']689 = the empirical values observed by Doty and Lundberg, +go", and by Elliott et at. (1956) for poly-L-leucine, +97", Tinoco and Woody (1960) point out that absolute magnitudes in this type of calculation depend upon the sums and differences of large numbers, so that agreement here may be fortuitous. Tinoco and Woody (1960) apply the same theory and spectral data to compute the rotatory contribution of light parallel and perpendicular to the helical axis, taking further into account the interaction of side chains with the helix. Inasmuch as their comparison of the predicted dispersion with empirical data, both for average dispersion and the difference between the parallel and perpendicular components, favors left-handed helices without, however, a definite indication of the complex nature of the dispersion, the capacity of this type of theoretical procedure to account for the rotatory behavior of helices and to resolve the issue of helical sense remains open to question. Efforts along these lines nonetheless continue, and the theoretical deduction of helical sense from dispersion data may yet be possible. I n this connection, Goodman (1961) has recently undertaken an evaluation of the interactions stressed by Moffitt et al. (1957) with results that appear promising, and he in particular emphasizes the critical role that series expansions play in any comparison of theoretical terms with empirical data expressed in the guise of phenomenological equations. In the absence of a valid correspondence between theory and observation, how may one regard the status of Eq. (18)? It is an explicitly phenomenological equation, that is, one developed for the purpose of expressing observed rotatory dispersion curves with a manageable number of constants. Once Xo is set, obedience to it is easily settled, and the dispersion of a large number of helical polypeptides show almost identical features when expressed by this equation. Its form has theoretical origins, but it can be demonstrated that this is not a consequence unique to helices. Kauzmann (19.57a) 1959) has shown that any interaction of a pair of identical chromophores results in splitting that will lead to a term containing (A2 - A:)+, and that upon consideration of interactions of this transition with different groups in the molecule, which give rotatory contributions with simple Drude dependence, an equation with the precise form of Eq. (18) is obtained. The generality of this form is also apparent from Murakami's treatment of the dynamical coupling of identical chromophores by the polarizability theory in which there is no explicit requirement for helical arrangement (Murakami, 1957), and his proposed phenomenological
+
OPTICAL ROTATION AND PROTEIN CONFORMATION
417
equation provides for complex dispersion in much the same way as does Eq. (18). Recent experiments (see Section 111, I ) which indicate that the dispersion of the p-structure of polypeptide chain can be described by Eq. (18) are thus consonant with these derivations and indeed imply that its form as such cannot discriminate between helical and extended conformations. The constants for the p-structure appear, however, to be characteristically different from those for helices; this clearly emphasizes that the empiricaI utility of this expression rests in large measure upon its calibration by known structures. Inasmuch as this form of rotatory dispersion is in principle characteristic of the interaction of like chromophores, one would wish to employ it in the investigation of ordered polypeptide structures. But, as will become evident in the experimental use to which Eq. (18) has been put, it possesses in addition the distinct practical advantage that Xo of helices is close to A, for disordered polypeptide chains, that is, about 220 mp, so that one equation can characterize mixtures of disordered chains and helices and at the same time yield a parameter, bo , uniquely related to helical content. This feature is highlighted by a lack of similar adaptability displayed by alternative empirical equations proposed to describe helical dispersion. Yang and Doty (1957) have fitted data for helical polyy-benzyl-L-glutamate to an abbreviated two-term Drude equation, [(Y]A
13.9 X
= --
8.0 (X2
- 0.2822)
yet helical poly-L-glutamicacid required a different set of constants. Sasisekharan (I 961) has employed an equation proposed by Chandrasekhar (1952),
to describe the dispersion of amino acids, polypeptides, and proteins. Although a single expression of this form suffices for random coils and appears as well to describe helical poly-L-glutamic acid, the dispersion of helical poly-7-benzyl-L-glutamate requires an additional abbreviated term. Furthermore, there is no uniformity among Xo values derived from this equation. Schellman and Schellman (1958) have suggested that the retention of the third-order term in the series expansion of the Drude equation will accommodate complex dispersion by an inverse cubic dependence upon (A2
- A:),
blx and indeed linear plots of
K'
= ___ X2
[C.]A(x2
- X,z + ( A 2
Kff - X33
- A:) against (1' -
are obtained for
418
PETER URNES AND PAUL DOTY
helical polymers when X, is in the vicinity of 120 mp. Save for the consideration that in Moffitt's treatment the average wavelength of the parallel and perpendicular components, X i in Eq. (16), of each interacting transition may be close to the wavelength of the Same transition in the absence of interaction and hence characterize its simple Drude dependence, there appears to be no compelling theoretical reason for the identity of the dispersion constant of the helix, Xo , with X, of the disordered chain. This is, however, an important empirical consequence of the inverse square dependence of (Az - A,") as it appears in Eq. (18). Although theory has as yet provided no satisfactory explanation in terms of electronic structure for the rotatory dispersion common to helical polypeptides, the question of why the simple dispersion of the peptide bond in the visible spectrum becomes complex as a helix is formed from a random coil can, in principle, be elucidated by direct measurements of rotatory phenomena at the electronic absorption bands concerned. Before we consider rotatory behavior at optically active absorption bands, it is possible, followingthe discussion of Moffitt and Yang (1956),to draw some inferences about changes in ultraviolet transitions from alterations in the visible spectrum alone. When a simple Drude equation is adequate to describe a set of dispersion data, this expression may be related through A, to the rotatory strengths, A ; , and wavelengths, X i , of the optically active transitions by the following definition,
As formulated by both Schellman (1958a) and Moffitt and Yang (1956), A: defined in this way permits the second term in a series expansion of the is thus an general Drude equation [Eq. (lo)] to vanish. The constant average of individual expressions to which contributions at high wavelength are heavily weighted because of the fourth-power dependence in the numerator. If the product A,Xi of one or more of these predominant long wavelength transitions should increase, either by an increase in Ai or X i or both, then A: must also increase its value in order that the second term in the series expansion remain zero and the dispersion simple. If the increases in some A;X; are sufficiently great, then no value of A% will effectively average out the discrepancies among the various products, and the simple Drude plot will become nonlinear. Consequently, the onset of complex dispersion may be formally attributed to the appearance of one or more strong rotatory bands at relatively long wavelengths. Since empirical evidence about the location of rotatory bands in helical polypeptides and proteins is just becoming available, it is important at this point to consider the salient facts about rotatory dispersion in regions of absorption.
OPTICAL ROTATION A N D PROTEIN CONFORMATION
419
D . Optical Rotation in Regions of Absorption: The Cotton E f e c t Thus far our discussion has centered upon rotatory dispersion in regions of relative transparency in which measurements are instrumentally feasible and theory reasonably secure. However, the featureless monotonic variation of optical rotation with decreasing wavelength changes radically at optically
OPTICAL ROTATION
hi
FIG.3. Idealized Cotton effect a t an isolated, optically active absorption band with its maximum a t X i . If the positive limb of the rotatory dispersion is t o the high wavelength side of the band, the Cotton effect is termed positive; if t h e negative limb is a t higher wavelength, t h e effect is negative. I n regions distant from the absorption band, the rotatory dispersion approaches simple Drude behavior.
active absorption bands. As an isolated optically active band is approached, the optical rotation will rise (or fall) sharply, turn and pass through the center of the band with a point of inflection, and again rise (or fall) as shown diagrammatically in Fig. 3. Associated with this strikingly different behavior of the rotatory dispersion, one also finds that the light emerging from the sample is no longer polarized in a plane but instead may be represented by an electric vector that describes an elliptical path (see Moscowitz, 1960a). Both the unusual dispersion and the ellipticity may be ex-
420
PETER URNES AND PAUL DOTY
plained by the unequal absorption of the right and left circularly polarized components of the beam, that is, by circular dichroism. Circular dichroism can be measured directly only with difficulty, for the expected differences in the extinction coefficient of right and left circularly polarized light are exceedingly small. I t can, however, be calculated from the ellipticity observed in the plane of polarization. It has become customary to refer to the distinctive rotatory dispersion alone as a Cotton effect, although the term as systematically used implies the presence of circular dichroism and ellipticity as well (Cotton, 1896; Kuhn, 1958). Any one of these three phenomena serves as a hallmark of an optically active transition, but it is the dispersion that is usually measured. Theoretical treatments of optical rotation in regions of absorption are able to provide a more complete picture of the contribution of a single electronic transition than does the Rosenfeld equation [Eq. @)I. They show in fact that each partial rotation not only approximates to a single Drude term in regions of transparency but also gives rise to an ideal Cotton effect a t the absorption band. Since the full rotatory dispersion for a molecule is the summation of its partial rotations, it may be envisioned as the superposition of many partial contributions, each with a Cotton effect a t its characteristic wavelength. These partial contributions hence combine far from the bands concerned to produce a dispersive tail fitting the Drude form. The treatment of Kuhn (Kuhn and Freudenberg, 1932) is based on a classic coupled oscillator model while that of Moffitt and Moscowitz (1959) proceeds from quantum mechanical considerations; both, however, lead to essentially the same result. If, in practice, a Cotton effect can be isolated, it may be characterized by tlhe wavelength of the active transition, and its rotatory strength can be estimated from rotatory parameters by methods that are discussed by Moscowitz (1960a, b, 1961). The correspondence of Cotton effects with ordinary absorption bands is not complete, for chromophores in symmetrical environments have no circular dichroism and hence possess optically inactive transitions. Conversely, optically active transitions may produce no detectable absorption and yet contribute strongly to optical rotation (Kuhn, 1958). Furthermore, Cotton effects rarely exhibit the ideal shape predicted by theory, for they are generally superimposed upon a background of other rotatory contributions that may make assignment of the active transitions questionable. Analysis becomes especially difficult if Cotton effects overlap to produce an irregular profile, one which is termed a multiple Cotton effect. Many examples of the complications that result may be found in the work of Kuhn (1958) and Djerassi (1960) and their coworkers. The latter have used Cotton effects associated with the carbonyl group in an empirical way to assign conformations of molecules containing
OPTICAL ROTATION A N D PROTEfN CONFORMATION
42 1
this chromophore, as in the steroid series. The resolution of active transitions can be facilitated by measurement of circular dichroism, which shares the localized character of ordinary absorption bands and further will specify the sign of the Cotton effect. Since electronic transitions can usually be assigned to specific chemical groups by absorption spectra, a similar correspondence can be made between Cotton effects and chemical constitution, but it must be kept in mind that the partial rotation associated with any given chemical group is not a property intrinsic to the chromophoric transition alone but reflects the spatial disposition of this group with respect to its neighbors. This is a matter of electromagnetic influence that is generally unimportant beyond 5 A. Despite the discrete character of partial rotations, their summation in the optical rotatory power of a molecule does not represent the additive effect of individual chemical groups hut is rather a most sensitive indicator of their mutual orientation. A Cotton effect is termed positive if it exhibits a maximum a t the long wavelength side of its absorption band and negative if it reaches a minimum (Fig. 3 ) . Since a Cotton effect is characterized by two extreme values of the optical rotation and three points of inflection, it technically qualifies as a type of anomalous dispersion defined by Lowry (1915, 1935). Lowry initially used the term “anomalous” to designate a variety of complex dispersion which passes a point of inflection, exhibits an extreme value, and then reverses sign as the wavelength is decreased. For example, both tartaric acid (Lowry, 1935) and poly-y-benzyl-L-glutamate in m-cresol (Fig. 1) display these features, and they may be accommodated in a two-term Drude equation by constants of appropriate sign and magnitude (see Todd, 1960). The use of ‘Lanomalous’’to describe a variant of complex dispersion is of importance primarily to remind one that anomalous features can appear in regions of relative transparency and hence should not be mistaken for an incipient Cotton effect. In order to avoid ambiguity, a Cotton effect may be designated as intrinsic anomalous dispersion (Heller, 1958). Djerassi and Klyne ( 1957) have proposed another nomenclature for rotatory dispersion curves in which those that do not display maxima or minima are called plain curves to differentiate them from those that contain Cotton effects, but this classification is of little relevance to polypeptide systems in which the fundamental distinction rests between simple and complex dispersion. The detection of optically active absorption bands is usually prevented by technical barriers, for they generally lie in the far ultraviolet region of the spectrum. Yet, largely as a result of instrumental improvements, the first measurements of Cotton effects associated with helical polypeptides have very recently been achieved. Simmons and Blout (1960) first measured a minimum in the rotatory dispersion of tobacco mosaic virus protein a t 232
422
PETER URNES AND PAUL DOTY
mp, and Simmons et al. (1961) have elaborated this suggestive finding by obtaining the ultraviolet dispersion of helical polypeptides and proteins. The data on poly-y-benzyl-L-glutamate in dioxane and poly-L-methionine
in methylene dichloride, solvents in which they are helical, show clearly that these dispersions exhibit minima at about 233 mp and maxima at about 220 mp (Fig. 4). Comparable data are not reported for aqueous solution, but the dispersions of poly-L-glutamic acid at pH 4.5 and native tropomyosin and paramyosin, which are known to be largely helical, also show minima at 233 mp of about the same amplitude, [m']233S -12,000". Ad-
OPTICAL ROTATION AND PROTEIN CONFORMATION
423
ditional measurements show that this effect virtually disappears in the ram dom coil of poly-L-glutamic acid and upon denaturation of the proteins, thus implying that the full deflection measured in nonaqueous solvents is a result of the helical conformation to which differences in amino acid composition contribute little. The shape of the dispersions in Fig. 4 suggests that the deflection is symmetrical about the point of inflection near 225 mp and that it hence describes the larger part of a negative Cotton effect centered at this point. If this is correct, then the Cotton effect arises from an optically active transition near 225 mp. Helical polypeptides in solution have no distinct absorption band here, but their extinction coefficient in this region is greater than that of random coils (Rosenheck and Doty, 1961), a phenomenon noted by Glazer and Smith (1960, 1961) in the difference spectroscopy of proteins at slightly higher wavelengths. Polarized absorption spectra upon films of oriented helical polypeptides have very recently revealed a weak transition near 222 mp that is polarized perpendicularly to the helical axis (Gratzer et al., 1961). The Cotton effect may thus correspond to this transition, one which may be an n -+ ?r* transition analogous to those found at 210 to 220 mp in simple amides (Ham and Platt, 1952; Peterson and Simpson, 1957). Even if a transition at 225 mp is the source of this Cotton effect, it can nonetheless be argued that the complex dispersion characteristic of helices in the visible spectrum involves additional transitions further in the ultraviolet. A single optically active transition giving rise to a negative Cotton effect will in principle contribute negative rotations at all higher wavelengths. If one assumes that the dispersion of the disordered chain represents the rotatory background upon which the dispersion of the helix is superimposed, then the more positive rotations of helical polypeptides in the visible spectrum imply that at least one transition with a positive Cotton effect contributes significantly to the visible dispersion. This possibility is in accord with formal analyses that derive complex dispersion from two closely placed transitions of opposite rotatory strength, as set out in the work of Moffitt (Moffitt, 191j6a; Moffitt and Yang, 19.56) and of Kauzmann (1957a, 1959). Simmons et al. (1961) point out that in the, absence of data at lower wavelengths, the entire rotatory deflection between 233 and 220 mp may be the positive limb of a sharp positive Cotton effect centered at some wavelength lower than 220 mp. Both this possibility and that concerning additional rotatory bands below 225 mp, as discussed in the foregoing paragraph, receive support from spectroscopic observations that implicate the peptide transition at 190 mp with the helical conformation. It has been established that the extinction coefficient of the peptide bond at 190 mp decreases from about 7000 to about 4000 upon formation of thc helix from
424
PETER URNES AND PAUL DOTY
the random coil (Imahori and Tanaka, 1959) and that a shoulder appears a t 205 mp (Rosenheck and Doty, 1961). Tinoco et al. (1961) have recently been able to give a theoretical account of this hypochromic shift based upon exciton splitting of the same transition. Furthermore, the splitting of this transition has now been directly observed by means of polarized absorption spectra from films of oriented helical polypeptides (Gratzer et al., 1961). A component near 190 mp is polarized perpendicularly to the helical axis, while that a t 205 mp is polarized parallel to it. I n the light of Moffitt’s general exciton treatment for the rotatory dispersion of helices, one would therefore expect these transitions induced by exciton splitting to have a fundamental influence on the rotatory dispersion of helical polypeptides. Until the dispersion is measured at 205 and 190 mp, the centering of the Cotton effect observed by Simmons et al. at 22.5 mp must remain tentative. However great the technical impediments may be, the investigation of the optical properties of helical polypeptides in the far ultraviolet promises to yield much information of both theoretical and empirical significance. Further work upon the optical activity of oriented helices, the study of which has been undertaken by Tinoco in a series of papers (Tinoco, 1957a, b, 1959), together with measurements of circular dichroism in this region may provide additional evidence of exciton splitting and permit estimates of rotatory strengths to be made. As will be seen, rotatory dispersion is a t present capable of detecting the presence of the 0-conformation and helices opposite to the standard sense only by gross variation in the Moffitt constants. Yet the discrete nature of rotatory dispersion a t absorption bands may well reveal features peculiar to the 0-form and, since L-amino acids in a right-handed helix may not completely cancel their rotatory contribution in a left-handed helix, give some basis for discrimination here as well. Despite the interest of ordered conformations, it is important that the rotat)ory dispersion of the disordered polypeptide chain not be neglected, for it not only participates in protein structure but the location of its optically active transitions in the far ultraviolet may also elucidate the ultimate origin of thc rotatory properties peculiar to helices and @-forms.
111. T H E OPTICAL liOTATORY nISPERSION OF SYNTHETIC I’OLYPI!PTII)~S Because of instrumental limitations, most optical rotatory measurements upon polypeptides and proteins have thus far been made in the near ultraviolet and visible regions of the spectrum, a range over which the Drude and Moffitt equations are valid. For this reason, attention will henceforth be directed to featureless curves of the type shown in Fig. 1. The immediate concern will be to describe the optical rotatory properties of synthetic polypeptides with respect to conformation in order that these attributes may be subsequently employed in the analysis of protein structure. Synthetic poly-
OPTICAL ROTATION AND PROTEIN CONFORMATION
425
peptides are appropriate models for proteins because both classes of molecule share the same backbone of peptide bonds. Since it is the disposition of the peptide bonds that chiefly governs their rotatory power, optical rotatory dispersion is a method appropriate for assessing the fundamental features which these polymers have in common. Synthetic polypeptides are, moreover, exceedingly useful models in that the relative simplicity of their side chains as compared to native proteins permits them to assume distinct conformations, the presence of which can be demonstrated by independent criteria, so that the rotatory properties of these conformations can be unambiguously measured.
A . The Establishment of Chain Conformation in Polypeptides The assignment of the precise conformation of polypeptide chains in solution is a difficult matter, for final judgment hangs on evidence gathered by a variety of methods, no one of which is definitive. X-ray diffraction is the sole method that can specify with certainty the relative positions of atoms within an ordered molecule and is hence capable of detecting a-helices, but this type of detailed steric analysis must be carried out in the solid state. Infrared spectroscopy provides a valuable bridge between the solid state and solution in terms of the hydrogen-bonding characteristics of helices and the extended or P-forms, yet it cannot distinguish with accuracy a polypeptide helix from its random coil. The burden of discriminating helical molecules in solution therefore falls upon a combination of light scattering and hydrodynamic techniques, both of which yield information on the shape and dimensions of macromolecules. The manner in which each of these procedures helps establish the presence of a conformation which can then be characterized by optical rotation will be briefly discussed. 1. X-ray Diflraction
With the development of practical synthetic methods for high molecular weight polypeptides, their study by X-ray diffraction became feasible. X-ray diffraction analysis is best carried out upon oriented, fibrous specimens which have a high crystalline content, for diffraction patterns reflect only those portions of a sample which have periodic order. Fortunately, pure, homogeneous polypeptides are in general crystalline and can be prepared in sufficiently high molecular weight to permit good fiber formation and ease of orientation. The first comprehensive studies of a synthetic polypeptide by X-ray diffraction were made by Bamford and his collaborators on polyy-methyl-Lglutamate (Bamford et al., 1951, 1952, 1953). These diffraction patterns were found by Cochran et al. (1952) to correspond closely to those predicted from the crystallographic model proposed by Pauling et al. (1951), a
426
PETER URNES AND PATJL DOTY
conformation designated as the a-helix. The right- and left-handed forms of this model for a polypeptide chain consisting of L-amino acid residues in their known configuration are pictured in Fig. 5. Strong evidence that this
I
1
FIG.5. Drawings of the left- and right-handed a-helical forms (to the left and right, respectively) of a polypeptide chain containing L-amino acids. The side chains, R , and hydrogen atoms attached t o the a-carbons in the main chain have positions corresponding to the known configuration of L-amino acids. (From L. Pauling and R. B. Corey, in Low and Edsall, 1956, p. 398.)
helix exists in poly-y-methyl-L-glutamate is provided by the intense reflection in the direction of the fiber axis with a spacing of 1.5 A, a feature initially observed by Perutz ( 1951) in poly-y-benzyl-L-glutamate. This meridional reflection corresponds to a translation per residue of 1.5 A along the axis of the a-helix. A second meridional reflection found at 5.4 A represents
OPTICAL ROTATION A N D PROTEIN CONFORMATION
427
the pitch of the a-helix, that is, the distance along the axis in which the helical chain makes a complete turn, one which contains 3.6 residues. The diffraction patterns observed by Bamford et al. (1954) for poly-L-alanine, the polypeptide most thoroughly studied by X-ray methods, failed a t first to correspond to calculated intensities for either right- or left-handed a-helices, as shown by Brown and Trotter (1956). However, on the assumption of hexagonal packing, Elliott and Malcolm (1956, 1958) have presented evidence that these patterns best fit right-handed helices, a conclusion that will be further discussed with respect to the bearing of helical sense upon optical rotatory properties. A water-soluble polypeptide, poly-Lglutamic acid, has recently been shown by X-ray diffraction also to exist in the a-helical conformation in the solid state (Johnson, 1959). The diffraction patterns displayed by helical synthetic polypeptides are similar to those first discovered by Astbury for fibrous proteins (Astbury and Street, 1931; Astbury and Woods, 1933), with the exception that a meridional spacing here occurs a t 5.1 A instead of 5.4 A. This particular pattern, which Astbury designated the a-pattern, may be reconciled with the a-helix if the helices are themselves coiled to form a cablelike structure, as suggested independently by Crick (1952, 1953) and by Pauling and Corey (1953). X-ray methods are able to distinguish helices from another hydrogenbonded conformation which occurs in the solid state, that of the extended or @-form.The diffraction pattern characteristic of this form, which displays a repeat distance of about 7 A along the fiber axis and perpendicular spncings of about 4.7 A, was observed in fibrous proteins by Astbury a t the same time as the a-pattern was discovered and was accordingly designated the P-pattern. The distinct structural connotations of these patterns, that the a-pattern represented a folded backbone while the @-patternwas associated with a n almost extended chain, were based on the reversible changes that appeared when fibrous proteins were steamed and mechanically stretched. Analogous transformations have been carried out by Bamford el al. (1951) on synthetic polypeptides, thus strengthening the conclusion that the X-ray pattern reflected the conformational state of the main chain. l’auling and Corey (1951) have proposed three models for the P-form, all of which involve polypeptide chains stabilized by intermolecular hydrogen bonds. One conformation consists of fully extended chains. In the other two, the chains are slightly buckled from full extension. The so-called parallel pleated sheet has its chains running in the same direction, while in the antiparnllel pleated sheet (Fig. S), adjacent chains run in opposite directions. It is sometimes difficult to make a choice between the latter two models on X-ray evidence, but the principal spacings observed in the @-patterncorrelate well with the general features of an extended chain in both the repeat distance of identi-
428
PETER URNES AND PAUL DOTY
cally disposed groups along it and the perpendicular interchain distance. Bamford el al. (1951) found that films of poly-y-methyl-L-glutamate cast from formic acid displayed a @-pattern. Although this same polymer cast from m-cresol or chloroform gives an a-pattern, the appearance of the Pform may be more a function of molecular weight than of solvent polarity. A detailed discussion of X-ray studies of synthet,ic polypeptides may be found in the monograph by Bamford et al. (1956).
FIG.6. The antiprtrallel pleated sheet structure of polypeptide chain?. (Marsh et al., 1955.)
2. Infrared Spectroscopu Infrared absorption spectra of synthetic polypeptides have provided iniportant ancillary evidence for the existence of a-helices and ext>ended0forms both in the solid state and in solut,ion. Ambrose and Elliott (1951a) showed that the chemical groups of the main chain that are capable of forming hydrogen bonds, the carbonyl and imino groups, exhibit characteristically different infrared absorption frequencies in the two forms. For example, the carbonyl stretching frequency (amide I band), one often used to assign conformation, is about 1655 em-' in the helical form and about 1630 cm-' in the extended form. They further found that these characteristic frequencies displayed maximal absorptions at definite angles of a plane of polarized radiation to the direction in which specimens had been oriented. Fibers known to be in the a-form by X-ray evidence showed maximum ab-
OPTICAL ROTATION A N D PROTEIN CONFORMATION
429
sorpt)ion for both the CO and NH stretching frequencies when the plane of polarization was parallel to the main chain direction, a finding which indicated that the valence angles of these groups were mutually aligned along the fiber axis as they would be in a helix. The p-form showed maximum absorption perpendicular to the chain direction, which is compatible with interchain hydrogen bonding. The frequency shift criterion for distinguishing these two forms gained immensely in value when further investigation established that it held for synthetic polypeptides in solution as well as in the solid phase, for it is impossible to use the X-ray criteria of conformation on solutions. Bird and Blout (1959) have more recently shown that dichroic differences can also be detected in solut.ions of helical polymers oriented by streaming, so that there is a complete correspondence between solid and solution for the helical and /3-conformations in terms of these distinctive infrared properties, and of these in turn with the primary X-ray criteria. As yet, however, infrared spectra cannot differentiate various helices that have been proposed (Donohue, 1953; Low and Edsall, 1956), nor is it clear that the random coil, which is in general the alternative conformation in solution, can be reliably distinguished from helices. Although the amide I frequency of helical poly-L-glutamic acid in DzO shifts from 1638 to 1644 cm-’ upon forming the random coil (Klemperer and Doty, 1960) and similar shifts are observed in copolymers of glutamic acid and lysine upon loss of helical content (Blout and Idelson, 1958), this carbonyl frequency can be almost identical in the two forms (Elliott et al., 19.5713, 1958). Additional techniques are therefore required to provide firm evidence for the a-helical conformat,ion in solution. 3. Light Scattering and Hydrodynamic Measurements
If synthetic polypeptides can exist in solution as long, rigid helices, then physical methods for measuring the shape and size of macromolecules should reveal their distinctive features. The dependence of the helical conformation upon solvent polarity as well as its rodlike character were established for high molecular weight poly-y-benzyl-L-glutamate by flow birefringence and the measurement of intrinsic viscosity (Doty et al., 1954, 1956). In dichloroacetic acid and trifluoroacetic acid, its flow birefringence was found to be negligible and the variation of intrinsic viscosity with molecular weight was that of a randomly coiled polymer. I n solvents of lower polarity such as ethylene dichloride, chloroform, and dimethyl formamide, the flow birefringence and molecular weight dependence of intrinsic viscosity were both high, indicating the presence of quite asymmet,ric, rodlike particles. It was shown that viscosity data could be fitted to Simha’s equation for the intrinsic viscosity of ellipsoids by assuming a constant minor axis of 18.3 A, which is equivalent to a cylinder 14.9 A in diameter. An a-helix of poly-y-benzyl-
430
PETER URNES AND PAUL DOTY
L-glutamate is theoretically 15.3 A in diameter, while X-ray evidence gives a value of 15.0 A (Arndt and Riley, 1955), so that this dimension of the rods is compatible with an a-helix. The length of rodlike particles will of course vary with molecular weight, but if they consist of a-helices with a length of 1.5 A per residue, then the mass-to-length ratio should reveal this proportionality. Light-scattering measurements upon samples of poly-y-benzyl-L-glutamate of different molecular weight in chloroform-formamide solution confirmed this expectation (Doty et al., 1956).Since a current report (Luzzati et al., 1961) indicates that somewhat higher values for the translation per residue have been found for poly-y-benzyl-L-glutamate by this method as well as by low angle X-ray diffraction, the importance of using samples of negligible or well characterized polydispersity must be stressed. The length determination by light scattering is a “z” average while the molecular weight is a weight average, so that the measured value for the translation per residue will increase with polydispersity. Consequently, it is relevent to note that measurements by Yang and Doty not yet published in detail (Doty, 1957) were carried out on samples of negligible polydispersity as demonstrated by flow birefringence and hence constitute more convincing evidence for the 1.5 A per residue value. Molecular dimensions in solution can also be computed from a knowledge of the molecular weight and rotatory diffusion coefficient, which has been obtained for poly-y-benzyl-L-glutamate in ethylene dichloride from dynamic electrical birefringence measurements (Tinoco, 1957b). The shear dependence of the intrinsic viscosity, which has been determined for polyy-benzyl-L-glutamate in m-cresol (Yang, 1958, 1959; Philippoff and Gaskins, 1959), also leads to a value for the rotatory diffusion coefficient. These studies uniformly indicate that poly-y-benzyl-L-glutamate in nonpolar solvents behaves as theory would predict for a rigid, asymmetrical particle, and moreover lead to lengths in good agreement with those calculated for corresponding 0-helices. These studies also show that this polypeptide in dichloroacetic acid is a random coil. The similarity of the dipole moment and dielectric dispersion of poly-y-benzyl-L-glutamate in dioxane and ethylene dichloride with properties expected for a-helices is a further point favoring their identity (Wada, 1958; 1959a, b, c), although the capacity of these essentially hydrodynamic methods to distinguish helices of slightly different, coordinates is doubtful. Systematic application of hydrodynamic arid light-scattering methods has been made in conjunction with optical rotatory studies upon poly-r-N-carbobenzoxy-L-lysirie (Applequist and Doty, 1961 ; Daniel and Katchalski, 1960, 1961) and poly-L-lysine (Applequist and Doty, 1961). Poly-r-N-carbobenzoxy-L-lysine in dimethyl formamide displays light scattering, viscosity, and flow birefringence behavior that is consistent with a predomi-
OPTICAL ROTATION AND PROTEIN CONFORMATION
43 1
nantly helical molecule containing some imperfections. The helix of this polymer is considerably less stable than that of poly- y-benzyl-L-glutamate, SO that short sequences of broken hydrogen bonds may well result from ordinary kinetic activity as well as hydrodynamic stress. Its helical nature in nonpolar solvents receives support from the characteristic infrared dichroism obtained upon samples oriented by streaming (Bird and Blout. 1959). Flow birefringence measurements on poly-L-lysine with progressive neutralization of its side chains at high pH indicate that it forms rigid rods with dimensions that correspond well to those of an a-helix. The hydrodynamic behavior of this polypeptide will be further described in connection with its optical rotatory properties (see Section 111. G , 3). Poly-L-glutamic acid has also been characterized by light scattering and hydrodynamic methods (Doty et al., 1957; Goldstein and Katchalski, 1960; Wada and Doty, 1961). Its viscosity increases as the side chains are neutralized by lowering the pH, behavior expected for the formation of a rigid rod, and its mass-to-length ratio at low pH as measured by light scattering is in good agreement with that of an a-helix. Two auxiliary techniques, electron microscopy and hydrogen-deuterium exchange, as well as a relatively new method, far ultraviolet spectroscopy, also provide useful data on chain conformation in synthetic polypeptides. Perhaps the most striking evidence for the existence of helices in solution has been obtained from electron microscopy of poly-y-bensyl-L-glutamate and poly-L-glutamic acid (Hall and Doty, 1958). Micrographs of these polymers sprayed onto mica from solutions in which their helical properties are established, ethylene dichloride and water a t pH 4.5, respectively, clearly show rods with diameters roughly those of a-helices and lengths of the same order as those expected for a-helices from molecular weight measurements in solution. The random coils of these polymers reveal no structure whatsoever upon similar visualization. The rate of hydrogen-deuterium exchange in synthetic polypeptides may be expected to reveal a characteristic difference between the helical and random coil conformations. The imino hydrogens of a flexible polypeptide backbone can be freely solvated, so that they will rapidly exchange with deuterium from appropriate solvents, whereas if they participate in the intramolecular hydrogen bonds of a helix, their rate of exchange will be much slower. Both poly-y-benzyl-L-glutamate in dichloroaceticacid (Elliott and Hanby, 1958) and poly-L-glutamic acid at pH 7.4 (Blout et al., 1961; Bryan and Nielsen, 1960) show rapid deuteration as measured by the characteristics of NH infrared bands, while under conditions promoting helix formation, chloroform and pH 4.5, respectively, the exchange is much slower. This method cannot distinguish among alternative intramolecularly bonded forms, that is, varieties of helices and extended structures. The marked hypochromism that synthetic polypeptides undergo at 190
432
PETER URNER AND PAUL DOTY
mp upon formation of the helix, a phenomenon described in Section 11, D, can also be employed as a criterion to distinguish helices from random coils. Unfortunately, this technique is limited principally to aqueous solut,ions with low salt content, for many ions, for example, chloride, and most organic solvents absorb in this region of the spectrum. Poly-L-glutamic acid and poly-L-lysine have been studied by this method (Imahori and Tanaka, 1959; Rosenheck and Doty, 1961; Tinoco et al., 1961) and it may well prove useful for the conformational investigation of water-soluble copolymers that serve as models for proteins. In summary, one may thus conclude from evidence provided by a variety of correlative methods that polypeptide chains assume a helical form in solution for clearly specified ranges of solvent polarity or pH. This result is in complete harmony with the structural proposal of l’auling et al. (1951) that polypeptide helices are stabilized by intramolecular hydrogen bonds, since competition for hydrogen bonds by polar solvents and electrostatic effects that influence this competition in water lead to disruption of these rodlike structures. Polypeptide helices of varying composition and different solvent requirements for helical stability appear, moreover, to have in common a specifically a-helical conformation that persists from the solid state into solution.
B. The Mofitt Parameters for Helical Polypeptides A sizeable number of high molecular weight synthetic polypeptides known to be helical in solution exhibit the dispersion parameters initially found by Moffitt and Yang ( 1956) for polyq-benzyl-L-glutamate and poly-L-glut,amic acid. As described in Section 11, C, obedience to the Moffitt equation requires an assignment of Xo through numerical trial, but in most instances no independent determination of this parameter has been made since linear plots have been found at once by using Xo = 212 mp. Given a value for Xo , several equivalent graphical procedures may then be employed to derive the coefficients of Eq. (18), a. and b, . Although Moffitt and Yang used [m’]k(Xz - Xi) and (Xz - Xi)-1 as the graphic variables, it is convenient to plot [m’l~(X~- X:)/X: against Xi/(Xz - A:), for one thereby obtains bo directly from the slope and a. from the intercept. A typical plot of helical dispersion is shown in Fig. 7 for a copolymer of 5 % L-tyrosine with L-glutamic acid at pH 4,along with that for the disordered chain at pH 7 (Urnes et al., 1961b). As the data in Table I show, bo values cluster about -630, so that the conclusion of Moffitt and Yang that X o and bo are relatively independent of solvent and side chain has been substantiated for a rather wide range of polypeptides. However, in a limited number of other instances different values of bo have been found, As will be discussed (see Section 111, F ) , t,hey may be seen as understandable exceptions and to that extent
OPTICAL ROTATION AND PROTEIN CONFORMATION
433
actually support the contention that a bu value of about -6630 is a direct reflection of the helical conformation. It is important to note that the value obtained for bo from a given set of data depends very strongly on the value assigned to Xo . This relationship may be most easily seen by referring to a Moffitt plot such as that illustrated in Fig. 7. If Xo is increased above 212 mp, then the term Xi/(Xz - A:) for any wavelength, X, that is greater than Xo will itself become greater and A , mp
0
0
600 500
I
/
400
I
I
I
2
4
340 I
I 6
300
I
I
I
8
10
I I 12
FIG.7. Graphical treatment by the Moffitt equation (18) with A D = 212 mp for rotatory dispersions of a synthetic polypeptide, a copolymer of 5% L-tyrosine with L-glutamic acid (PTGA). Helical form a t pH 4.0 in 0.1 M phosphate buffer, .--o. Random coil at pH 7.0 in the same solvent, 0--0. (Umes et al., 1961b.)
its reciprocal, (A2 - Xi)/X:, smaller. Points on the Moffitt plot corresponding to a given set of values for [m’]~ will become spread out along the abscissa and contracted on the ordinate, therefore making the slope of the plot, bo , less steep. Thus the absolute magnitude of bo will vary inversely with Xo . This dependence of 6 0 on XO is not trivial. Downie et al. (19ri7) report, for example, that bo for the same dispersion data of poly-L-leucine changes from about -470 to -640 upon altering Xu from 212 to 200 mp. As in this instance, dispersion data are usually not sufficiently precise or do not extend over a sufficient spectral range to permit independent assignment of Xo , so
TABLE I inthetic Polypeptides with Standard Rotator Properties*
--
Polypeptide Poly -L-alanine
Poly-p-benzyln-aspartate
Poly-y-benzylL-glutamate
Solvent
Optical rotation
ao
-
CHClr , 99%; DCA, 1% TFA CHCla ,70%; DCA, 30% TFA CHCls
-
-426 -100 -631
DCA
-
DCA EDC
+ZOO -636
Dioxane
-630
CHCls
-625
DMF
-666
1:4 EDC:DCA
-630 -
E DC
-
DCA
-
Dioxnne TFA E DC Hydraeine DMF Dioxane CHCla
-
-635 0 -620
-682 -576
DCA
Poly-c-N-carbobeneoxy-i-lysine Poly-L-glutamic acid
Reference
bo
0
CHCla Dioxane DMF
-670 -670 -625
CHCls CHCls ,SO%; DCA, 50% DMF
-540 0 -580
1:2 Dioxane:HsO, 0.2 M NaCI, PH 4.72
-625
1:2 Dioxane:HaO, 0.2 M NaCI, p H 6.56
-
HO, 0.2 M NaCI, p H 4.5 H10,O.Z M NaCI, p H 8
-
HzO, p H 4.4 HO, p H 10.5 1 :5 2-Chloroethanol: HSO, 0.1 M RbCl, p H 5 1:5 Z-Chloroethanol: H a , 0.1 M RbCl, p H 7.5
-610 +50
-
-
434
Downie e t al. (1957) Downie et al. (1967) Fnsman (1961a) Faaman (196la) Blout and Karlson (1958) Blout and Karlson (1958) Karlaon et al. (1960) Moffitt and Yaw (1966) Moffitt and Y a w (1956) Moffitt and Yane (1956) Moffitt and Yaw (1956) Moffitt and Yang (1956) Yang and Doty (1957) Yang and Doty (1957) Blout (1960) Blout (1960) Blout (1960) Yang and Doty (1957) Mitchell et al. (1957) Mitchell et al. (1957) Blout and Karlson (1958) Blout and Karlaon (1958) Karlson et ol. (1960) Simmons et al. (1961) Applequist and Doty (1961) Fasman (1961a) Fasman (196la) Moffitt and Yang (1956) Moffitt and Yeng (1956); Doty et al. (1957) Doty e l al. (1957); Yang and Doty (1957) Blout and Idelson (1958) Blout and Idelson (1958) Blout (1960) Blout (1960) Goldstein and Katchalski (1960) Coldstein and Katchalski (1960)
435
OPTICAL ROTATION AND P R O T E I N CONFORMATION
TABLE I-Continued Polypeptide
Solvent
Poly-L-glutamic acid
HIO, 0.1 M NaCl, p H 4.5 Hn0,O.l M NaCl,pH8.0 Dimethyl sulfoxide H a , 0.18 M NaCl, acetate buffer, p H 4.75 Benzene TFA CHCls, 70%; TFA, 30% TFA H a , 0.2 M NaBr, p H 11.1 Ha,0.2 M NaBr, p H 6.1 H a , 0.2 M NaBr, p H 11.9 Hn0, p H 6.8
Poly +leucine
Poly-L-lyaine
Copoly-L-lysineL-glutamicacid (equimolar) Poly-L-methionine Poly-y-methyl-& glutamate Copoly-L-tyrosyl-L-glutamic acid, 5% tyrosine
Optical rotation
bo
Reference
-
Wadn (1960) Wada (1960) Fasman (196lb) Simmons et al. (19131)
-560
-600 -435 -125
-
-650 0
Downie et al. (1957) Downie et al. (1957) Fasrnan (19618) Fasman (1961a) Applequiat and Doty (1961) Applequist and Doty (1961) Applequist and Doty (1961) Applequiat and Doty (1961) Doty et aE. (1958)
2-Chloroethanol
-636
TFA CHCh
-630
Doty el al. (1968) Fasman (19618)
TFA, 80%; DCA, 20% Methylene chloride DMF
0 -630 -544
FBsman (19618) Simmons el al. (1961) Goodman ct al. (1961)
+50 +68
Goodman el al. (1961) Goodman el al. (1961) Urnea et al. (196lb)
$45
Urnes el al. (19Blb)
DCA TFA H a , 0.1 M POI buffer, pH 4.0 H a , 0.1 M Po4 buffer, p H 7.0
0
-615
All measurements of ao and bo are based on AO = 212 m+. Solvent abbreviationix CHCls , chloroform; DCA, diohloroacetic acid; DMF. dimethyl forrnarnide; EDC, ethylene dichloride; TFA, trifluoroacetic acid. NL: nonlinear plot.
that the value of 212 mp is almost always assumed. As a characteristic of the complex dispersion of helical polypeptides, Xo = 212 mp has primacy only because there have been no serious contenders in the usual wavelength range of measurement, 35MOO mp. lo is that wavelength which allows higher order terms in a series expansion of Eq. (17) to be neglected (Section 11, C), with the consequence that the phenomenological two-term Eq. (18) can describe a given set of data in linear fashion. The function of Xo with respect to the Moffitt equation is thus analogous to that of A, for the simple Drude equation. Since Moffitt and Yang report a latitude of &5 mp in the choice of Xo ,other values near 212 mp may serve this function equally well. However, for purposes of comparing bo values among
430
PETER URNER A N D PAUL DOTT
polymers and especially in the use of bo for determining part,ial helical content, it is essential that t,he same value of Xo be used. As may be seen in several scattered examples, the optimal value of XO may shift if wider ranges of data are to be accommodated. A linear Moffitt plot for poly-y-benzyl-L-glutamate in chloroform using XO = 212 mp has been obtained for the range 237-578 mp with a bo of -660 (Karlson et al.,
t
\ \
-3000
FIG.8. Moffitt plots at two different values of XO , 212 and 216 mp, for the dispersions of a copolymer of 501, I.-tyrosine with L-glutamic acid (PTGA) from 700 mp down t o 240 mp. The plots based upon 212 mp are an extension of the visible and near ultraviolet data represented in det.ail in Fig. 7 . Helical form in 0.1 M phosphate buffer, pH 4: ha = 212 mp, A - A; XO = 216 mp, O--O. Random coil in same solvent, pH 7.0: Xo = 212 m p , A - A; X O = 216 mp, 0-0.(Urnes el al., 1961a.)
1960), but in another solvent, ethylene dichloride, a Xo value of 208 mp was required to make the plot linear down to 248 mp (Savitz and Doty, 1958). The dispersion for a helical copolymer of 5 % L-tyrosine with Lglutamic acid in 0.1 A!! phosphate buffer a t pH 4.0, the visible and near ultraviolet portion of which is represented in Fig. 7, requires a Xo of 216 mp to bring the data between 240 and 280 m p into line with measurements a t higher wavelengths (Fig. 8). As XO is altered from 212 to 216 mp, bo derived from visible and near ultraviolet data for this polymer changes from -615 to -535. An alteration to 218 mp is required to correct for a similar negn-
OPTICAL ROTATION A N D PROTEIN CONFORMATION
437
tive curvature in the Moffitt plot down to 240 mp for the dispersion of polyglutamic acid a t p H 4.25 in 0.25 M NaCl (Klemperer and Doty, 1960). Replotting of the visible and near ultraviolet data in this case changes Do from -635 to -535. This latter finding shows that the curvature for copoly-L-tyrosyl-L-glutamic acid does not arise from tyrosyl absorption bands, although the difference between 216 and 218 mp may reflect the difference in solvent. Since Xo can be specified to within f l mp with the aid of low wavelength data, further measurements upon water-soluble polypeptides in this spectral region may indicate that a dispersion constant other than 212 mp should be consistently employed in the Moffitt equation. As in these instances, any systematic change in Xo will produce a new value of b0 to characterize helical dispersion. It is inevitable that curvature will set in as optically active absorption bands are entered, but in view of the striking changes in the dispersion below 240 mp (Fig. 4), it is surprising that the equation holds as far as it does. From the same linear plot by which a value for Xo is shown to be appropriate for helical dispersion, the intercept a0 can be obtained as well as the slope bo . Although a. varies both with the polypeptide concerned and the conditions under which it is helical (Moffitt and Yang, 1956) and for this reason cannot be given a constant value to characterize helical dispersion in general, it can serve as a parameter for any single set of data. If Eq. (18) truly describes a given complex dispersion, its form shows that these three parameters, Xo , D o , and uo, are sufficient to specify and indeed reproduce the dispersion. From a formal point of view, however, there are four possible candidates, the foregoing three together with the variable [m’lx, specified a t some particular wavelength. Since the existence of some value of Xo that yields linear plots is a necessary test for the applicability of Eq. (18) to a set of data, Xo is the fundamental parameter of Moffitt dispersion and accordingly must be stated. The constant Do is likewise always measured, for it is the coefficient of the second term, the one which permits Eq. (18) to describe complex dispersion by virtue of its inverse square dependence upon (Az - X i ) . The constant bo specifies the extent to which t,his second term participates in the dispersion and thereby provides a direct measure of the complexity. Of the remaining two candidates, either the coefficient a. or [m’]xwill suffice to complete the description of the dispersion, although [m’]~ is more often recorded. It is, however, desirable to report numerical values for bot)h of these latter constants, for, as is true of the simple Drude equation (Section 11, B ) , the measured rotation a t conventionally high reference wavelengths may be somewhat discounted in the graphical treatment (see Fig. 7 ) with the consequence that [m’Ixmay not be precisely equivalent to the intercept uo . In summary, then, the optical rotatory dispersions of many synthetic L-
438
PETER URNES AND PAUL DOTY
polypeptides known by independent evidence to be helical can be expressed in linear form by the Moffitt equation with Xo = 212 mp. The helical dispersions display a further uniformity in the relatively constant slope, bo , of a Moffitt plot, with values near -630. The values of the intercept a0 and the rotation [m’]x do vary from one helical polypeptide to another, but these constants, toget,her with XO and bo , complete the characterization of any single set of data by the Moffitt equation.
C. The Rotatory Contribution of the Helix If one compares the dispersion of a helical polypeptide with that of its random coil (Fig. 1), it is natural to conceive of their difference as originating in the rotation of the helical form. From this point of view, the effect of the helix is superimposed upon the intrinsic rotatory power of a disorganized polypeptide chain in much the same way that the dissymmetry of Pasteur’s spiral staircase is superimposed upon its component steps (see Section 11, B ) . The rotatory contribution of this dissymmetrical structure is itself of interest, and its characterization in fact shows that dispersive parameters in addition to bo and XO may be used to detect its presence. Two types of procedure have been used in the attempt to isolate this contribution. Both of these recognize that the asymmetry inherent in amino acid residues possessing side chains also contributes to the observed helical dispersion and therefore must in some manner be subtracted from it. In the first, the dispersion of the disordered chain is subtracted from the dispersion of the helical polypeptide and the difference interpreted as a direct expression of the helical form. In the second, amino acid residues of opposite configuration are introduced into a helical polypeptide so as to approach a meso-helix in which intrinsic residue contributions to the rotation cancel one another. As we shall see, a meso-helix, which is defined as a helix of single sense containing equal numbers of L- and D-residues, cannot in reality be formed because of steric hindrance among side chains. To obtain the dispersion of the helix itself, therefore, the rotatory properties of the meso-composition must be reached by extrapolation from polymers with smaller proportions of D-residues that interfere less with helical stability. Before discussing these attempts to isolate the rotation of a helix devoid of side chains, it is important to acknowledge the extent to which a n exercise such as this is limited. It is difficult to avoid referring to the helix as though it were an entity quite separate from the side chains, both because of structural metaphors like “framework,” “core,” “backbone,” and “skeleton” that have been used and because theories of helical dispersion, by focusing on t,he peptide bonds as the source of its peculiar features, lead one to think of the helix itself merely as an array of peptide bonds. Kauzmann (1957a) has, however, quite correctly pointed out that a simple
OPTICAL ROTATION AND PROTEIN CONFORMATION
439
partitioning of sources of optical rotatory power is at variance with theory, so that in any real dispersion the mutual effect of all groups that can interact must be taken into account,. This proviso should forestall any facile interpretation of efforts to obtain the dispersion of the helix itself by experimental subtraction of discrete chemical groups. 1. The Comparison of Helical Dispersion with That of a Disordered Chain
The simplest way of isolating the dispersion of the helix is a straightforward subtraction of the dispersion of the disordered chain from that of the helical polypeptide. I n order to carry out this subtraction, however, both simple and complex dispersion must be cast in the same mathematical form. The relation between these two types of dispersion and the manner in which they may be assimilated to one another will therefore be examined in some detail, for the results will be essential not only to the question a t hand but also to a quantitative consideration of the central problem posed at the outset of this review, the dispersion of a mixture of helically arranged and disordered regions. Aside from the helical form, the principal source of optical activity in a helical polypeptide lies with the asymmetric carbon atoms of individual amino acid residues, so that it is these intrinsic rotations that one wishes to eliminate to reach the dispersion of the helix itself. For this purpose, a plausible case can be made for obtaining the intrinsic residue rotations from the rotatory properties of the disordered chain. Despite rotations of differing magnitude near the ends of chains, the mean residue rotation of a random coil with a single species of residue will approach a constant value as the chain length is increased. This constant value may therefore be equated with the rotatory power of an “interior” residue (Doty and Geiduschek, 1953; Schellman and Schellman, 1958). The rotatory power of a randomly coiled polypeptide chain containing more than one species of amino acid can furthermore be computed on the basis of additive contributions of its constituents (Schellman and Schellman, 1958), and the rotatory cancellation of L-residues by D-residues of the same species is a linear function of composition (Elliott et at., 1956; Downie et al., 1957), thus gixkg support to the view that the mean residue rotation of the random coil reflects the rotations of individual residues which have no net mutual interaction. The mean residue rotation of a randomly coiled chain is known to be sensitive to solvent, but in a given solvent it is possible to regard this value as a direct reflection of the intrinsic residue rotation to which solvent interaction contributes a constant amount. If the spatial orientation of the side chain to its asymmetric center is fixed, the intrinsic rotation will be a constant, and inasmuch as models of a-helices can be built in which side chains undergo no steric strain, one may suppose that this
440
PETER URNES AND PAUL DOTY
value will remain the same as the residue is incorporated into a helix. On this rationale, then, the dispersion of the random coil can be suhtractcd from that, of the helical polymer to arrive at the dispersion of the helix itself. The polypeptides in Table I all exhibit simple dispersion in the random coil, so that their optical rotation is given by the simple Drude equation at any wavelength within the range of its validity,
The constant A in Eq. (11) is here written aAE , thc superscript D drsignates a disordered form, and a D is the rotation Coefficient for this conformation. Dispersions for polypeptides in helical form, H , fit the Moffitt equation,
so that difference between them at any wavelength will on this basis give t,he dispersion of the helix alone.
The A, values of many random coils are in fact close to Xo , 212 mp. If A, is assumed to equal A. , then this difference reduces to a two-term equation, thus assimilating the simple dispersion of the disordered chain to the complex dispersion of helical polypeptides.
This expression, which purports to characterize the helix alone, suggests that it has complex dispersion of the Moffitt type characterized by bo , A. and a third constant,, ( a f - u D ) as , well as making a definite rotatory contribution at each wavelength, A[ml]f-”. a. b f and A0 . If this characterization is adequate, then bo should disappear completely with the random coil, and a test for this adequacy is to plot the data for the random coil in Moffitt form to determine if this coefficient is indeed zero. It is zero in many cases, but bo does not vanish for some polypeptides that can be shown by independent evidence to be completely disordered. Random coils of poly-L-glutamicacid (Blout, 1960) and numerous copolymers of it (Friedman and Doty, 1961), consistently give bo values of about +50, while bo of the Moffitt plot for the random coil of copoly-~t,yrosyl-L-glutamicacid pictured in Fig. 7 is +45. Poly-L-alanine and polyL-leucine undergo a sharp conformational transition from the helical form
OPTICAL ROTATION AND PROTEIN CONFORMATION
44 1
as the concentration of trifluoroacetic acid in chloroform is increased, and yet bo in 100 % trifluoroacetic acid is about - 100 for both polymers (Fasman, 1961a). Poly-P-benzyl-L-aspartate in dichloroacetic acid gives a bo of about -200 and its enantiomorph a value of about +200 even though both forms are random coils in terms of viscosity measurements and the conformational transitions that occur at smaller proportions of dichloroacetic acid (Bradbury et al., 1960b; Karlson et al., 1960). The origin of nonzero bo values in these cases is not entirely clear, but it can be formally traced to the difference between XO and X, values for the simple dispersion of random coils and hence to a failure of the assumption that X, equals Xo . If A, equals Xo , then the first term of the Moffitt equation will of course be the same as the simple Drude expression known to describe the data and, there being no necessity for a second term, bo will vanish. However, if X, differs from Xo , the MoEtt plot may still be linear but with a nonvanishing slope. Thus dispersion data that are simple when referred to one dispersion constant may appear complex when plotted against another by a form that sees matters as complex, thereby generating what may be properly suspected as “pseudocomplexity.” The Moffitt equation was initially intended to describe the complex dispersion of polypeptides for which the simple Drude equation is inadequate, but, as will be seen, its form is also applied to protein dispersions which can be expressed equally well by either formula. I t is therefore important to examine more fully the relation of the two equations for cases in which both fit the data. The general relation of X, to the parameters of the Moffitt equlttioii has been stated by Downie (1960). If a given set of dispersion data obey the simple Drude equation, A, may be obtained from the slope of [m’]xX2plotted against [m’lx , d([m’]xX2)/d[rn’]x(see Section 11, B ) . If each value of [m’Ix can also be accommodated by the MoEtt equation, a condition which is often satisfied for measurements in the spectral region 350-600 mp, then Eq. (18) can be substituted into this slope and the appropriate differentiation carried out with the following result.
Under these conditions, then, A, is a function of A0 and the ratio bolao as well as the wavelength range, represented by values of X, in which measurements are made. If bo/ao is zero, then A, = X O . Alternatively, it can be shown by numerical trial that for a set of data in the usual wavelength range, 350-600 mp, and with Xo set to 212 mp, A: will be constant for ratios
442
PETER URNES AND PAUL DOTY
as high as +0.6 and as low as -0.2. It will be greater than A: for positive ratios and less than X i for negative ratios. Thus, if A, for a simple dispersion is unequal to Xo , treatment of the same data by Moffitt form will lead to a nonzero value of bo . Large deviations of A, for randomly coiled chains from A0 of 212 mp have in fact been observed in a number of cases. For example, polyy-benzyl-Lglutamate in hydrazine has a A, of 210 mp, but in dichloroacetic acid it becomes 180 mp (Yang and Doty, 1957). Treatment of the data for poly-ybenzyl-L-glutamate in dichloroacetic acid by the Moffitt equation will therefore produce a nonzero value for bo , although it is not possible to specify in advance its magnitude or sign, for these depend upon the intercept, a. . A, for the random coil of copoly-L-tyrosyl-L-glutamic acid represented in Fig. 7 is 209 mp, while bo is +45, so that in this instance a small difference between X, and Xo is sufficient to give a noticeable bo value. The data of Goodman et al. (1961) for poly-y-methyl-L-glutamate and of Blout (1960) for poly-L-glutamic acid also reveal that positive bo values are associated with X, values less than 212 mp, as shown in Table I. Since there is independent evidence that these polypeptides are completely disordered, an interpretation of nonzero bo values as signifying helical content is obviously in error. These polypeptides nonetheless present a problem for characterizing the dispersion of the helix itself. If X, for these disordered chains does not equal Xo , with the consequence that bo does not vanish when the helical form is destroyed, should not the foregoing derivation, based on the equality of X, and Xo , be rejected? It will be retained for reasons that are discussed below, but let us first consider an alternative device for assimilating simple dispersion to the Moffitt form, one which circumvents this assumption. What can be adequately represented with two constants, A, and aD, can equally well be described with three, Xo , uf, and b f . Consequently, the reduced mean residue rotation for the disordered form may be written
As long as one knows that these parameters refer to an established conformation, the disordered chain, this redundancy or pseudocomplex description is innocuous, and it does permit a direct comparison of coefficients characacterizing the disordered and helical conformations by virtue of a mathematical form common to both. If b f has a value other than zero, then taking the difference between [m’]:and [ml];, given respectively by Eqs. (24) and (28), produces a coefficient (# - bf) for the helix itself, one which may be designated b f - D . Precise studies of given polypeptides for which X, of the disordered chain does not equal Xo should therefore be
OPTICAL ROTATION AND PROTEIN CONFORMATION
443
based on b t - D as a constant corresponding to a complete helix. For these cases, any variation of A, and b f with change of solvent must be explicitly taken into account. A derivation based on the approximation that A, equals A. and its corollary that bo of the disordered form is zero is, however, both reasonable and advantageous, particularly for the structural interpretation of the rotatory dispersion of proteins. Values of A, for standard random coils in aqueous solution and for denatured proteins do not differ widely from A 0 , and bf appears to be close to zero in the majority of cases in which it has been measured. The A, values observed generally fall between 210 and 230 mp, but it is difficult to specify the sources of this variation. As shown in Table I, poly-y-benzyl-L-glutamate, poly-y-methyl-L-glutamate, and poly-L-glutamic acid illustrate that A, for random coils can vary with the disordering solvent. Correction for the dispersion of refractive index of the solvent will change A, to some extent (Tinoco, 1959), but it is presumably dependent upon specific solvent effects as well. I n his study of the disordered proteins clupein and the oxidized A chain of insulin, Schellman (1958e) has found that A, depends upon the ionic species in aqueous solution, although it varies little with ionic strength. It also appears to be essentially independent of urea, pH, and temperature, all typical denaturing agents. That A, is also a function of amino acid composition is shown most strikingly for synthetic polypeptides with unusual rotatory attributes (Table IV). In the absence of further systematic investigation upon chains known to be completely disordered, one cannot distinguish compositional differences from variation in solvent conditions as giving rise to the range of A, values that have been reported. Furthermore, the actual quantitative effect upon bo of a difference between A, and A,, is documented in only a few instances. In the cases of copoly-L-tyrosyl-L-glutamic acid and poly-y-methyl-Lglutamate cited above, the effect is sizeable. However, as shown by Eq. (27), it is the rat,io bolao rather than bo itself that is a function of this difference. For a given ratio, if a. is small, then bo is likewise small, a possibility that may account for the negligible values of b, with wide variation in A, of poly-0-acetyl-L-serine and poly-L-serine (Fasman and Blout, 1960; Table IV). If we are primarily concerned with setting up a standard case for application to proteins of unknown conformation rather than a general treatment which will accommodate all exceptions, then because of both the closeness of A, for disordered chains to A 0 and the imponderable factors in assessing the small deviations that do occur, it is reasonable to incorporate this approximation into the standard case. As will be seen with greater cogency in the derivation of the dispersion for a mixture of helices and random coils (Section 111, G, 2), a pattern of analysis based on the equality of A, and A 0 has, in addition, the advantage that ba becomes a unique measure of partial helical content. Since bo for the
444
PETER IJRNES AND PAUL DOTY
disordered chain is assumed in the ideal case to be zero, an estimate of helical content can be made from the magnitude of this parameter for the ordered or native forms alone. In contrast, values of helical content obtained from the intercept, a. , and the rotation at a single reference wavelength, [rn’]~, require measurement of the disordered forms as well. Since cases may arise in which protein chains do not completely unfold under denaturing conditions so that one cannot obtain the disordered state for reference purposes, the possibility of a direct assessment in the native form is attractive. It is true that this direct estimate of helical content will be in error if the A, characteristic of disordered regions in the native structure differs from Xo , as will be further discussed (Section IV, D),but it seems preferable at the present stage of our knowledge to be able to make a definite, if approximate, evaluation of helical content than to refrain from a quantitative statement. For these reasons, the derivation based upon the approximation that A, equals Xo will be retained. As a consequence for the question at, hand, the dispersion of the helix iteslf, one can therefore generalize that the coefficient b f with a value of about -630 appears to be n property of the isolated helix. It is, of course, the merit of the Moffitt form that its A 0 for polypeptide helices is close to X, of their disordered states, thus permitting simple dispersion to be incorporated with complex dispersion in a straightforward way and giving the parameter bo a singular role. We have seen that this equation is also capable of describing simple dispersion for which X, does not equal Xo , with the result that bo has a value other than zero. On grounds of mathematical convenience, it can thus be used directly to represent the dispersion of disordered chains. If, however, a conformation which gives rise to simple dispersion is not known with certainty, as is the case for globular proteins, then the application of the Moffitt equation to these data must receive further justification. This will be attempted for globular proteins after their dispersions are described (Section IV, C). As a check on the use of this equation, it is always advisable to test a set of dispersion data for adherence to the simple Drude equation, both to detect genuine complexity and to identify simple dispersion that is susceptible to misinterpretation by a complex form. b. (a: - a D ) . The expression proposed for the dispersion of the isolated helix, Eq. (26)) suggests that a constant involving the intercept a: is a further characteristic of this structure. In order to assess whether the difference (a: - a ~ is) in fact as invariable as b:, one must measure the respective coefficients, a D and a t , of Eqs. (23) and (24). Although a D will be an appropriate measure for the disordered form if the assumption underlying Eq. (25)) that A, equals Xo , is justified, it is preferable in this inst,ance to suhstit,ut,ea,” of Eq. (28) for an. Although a: and a” will in
OPTICAL ROTATION AND PROTEIN CONFORMATION
445
most cases have almost the same magnitude, the calculation of a: by means of the Moffitt equation permits a more direct, comparison with a! to be made. The difference between and a:, which purports to be a const,ant, may then be represent’ed by a single term, a:-”.
at
ateD = a: - a t
(29)
Constants which interpret a: and but are operationally equivalent, to them have been introduced on an a priori basis into Eq. (18) for the dispersion of a helical polypeptide (Doty, 1957, 1958, 1959). As has been suggested, it is reasonable to think that the dispersion of a helical polypeptide arises from two principal sources, the intrinsic asymmetry of individual residues and the dissymmetry of the helical form. The coefficient of the first term, a. , may therefore be regarded as the sum of two coefficients, one representing the contributions of individual residues, a t , and the other, a:, all interactions peculiar to the helix that have, like the residues, ordinary wavelength dependence, (A2 - At)+. On tJherationale delineated above, that the rotatory properties of the disordered chain may be taken as representat,ive of intrinsic residue rotations, the measured coefficient a: may be usefully interpreted by equating it with a t . If conditions in any given case warrant this interpretation, then a: may be subtracted from the observed intercept for the helical polypeptide, a. , to obtain the coefficient of the helix alone, a:. At this point a conflict in notation must be acknowledged, for the expression a: has been used both for a n a priori constant characteristic of the helix alone and, in Eq. (24), as a purely descriptive symbol denoting a specific value for the parameter a. given by the dispersion of a helical polypeptide. Since it now appears more consistent t o reserve uf for this latter capacity, the constant peculiar to the heIix will, in this review, be designated by atwD.As indicated in its definition, Eq. (29), a value for a:-D is obtained in exactly the same way as values have been determined for u: in the former notation. The coefficient a: is quite sensitive to solvent, so that estimates of the intrinsic residue coefficient, a t , derived from it will vary with environment. Since a: is a useful measure only if it is effectively const,ant for both the random coil and the helical polypeptide, one would wish to measure a: of the random coil and a: for the helical dispersion under identical solvent conditions. Inasmuch as solvent changes can affect A, and thus influence a: , comparable measures of a:-” may be expected only for polypeptides that have similar X, values when disordered, which for convenience may be stipulated to be close to Xo . These severe requirements are met principally by ionic, water-soluble polypeptides in which there is little solvent change over the helix-coil transition and in which A, s X o . Poly-L-glutamic acid and poly-1,-lysine, which
446
PETER URNES AND PAUL DOTY
appear to meet these conditions at an ionic strength of about 0.2, yield a value for a k D of about +650 (Doty, 1958). Yet even under these favorable circumstances, the value of a:-” remains dependent upon the ionic strength of the solvent. As will be seen in the next section, [ a ] of ~ ,these ~ ~ polypeptides in the disordered form can vary by as much as 25” with ionic strength. This variation influences a F Dprimarily through its effect on a: rather than u t , for rotations characteristic of the helical form appear in these cases to be less sensitive to environment. For example, poly-L-lysine in water gives an of 4-950 (Applequist and Doty, 1961). It is perhaps a coincidence that equimolar copoly-L-lysine-L-glutamic acid in a 2-chloroethanol-trifluoroacetic acid solvent system yields a similar high value for a:-”: 870 (Doty et ul., 1958). c. A[m’]f-”.The change in optical rotation on going from helix to random coil, A[m’]f-D,at a given wavelength is an unreliable index of the helical contribution, for although it is uniformly in a levorotatory direction in the visible spectrum for polymers in Table I, it varies from 40 to 145” a t 589 mp, the sodium D line most often used for comparative purposes. The principal source of this variation is undoubtedly the sensitivity of the random coil to solvent interaction, so that the magnitude of A[m’]f-”is a function not only of the helical contribution but also of the particular solvent in which the chain is disordered. For example, the random coil of poly-ybenzyl-L-glutamate in dichloroacetic acid exhibits [m’]689of - 26.5”; in hydrazine, -52.5”; and in trifluoroacetic acid, -73.7” (Yang and Doty, 1957). Transitions that can be brought about without substantial changes in solvent, as by altering the temperature or pH, are thus more likely to represent the effect of the helical conformation. Values for A[m’]%fobtained from ionic polypeptides in aqueous solution are in fact more uniform and are in general about go”, lying between 0” for the helix and -90” for the random coil. This estimate does, however, depend upon ionic strength. The disordered forms of poly-L-glutamic acid (Idelson and Blout, 1958; Wada, 1960; Wada and Doty, 1961) and poly-Llysine (Applequist and Doty, 1961) become less levorotatory at 589 mp by as much as 25” as the concentration of a given salt increases, whereas the helical forms display considerably less variation. The rotation of disordered poly-L-glutamic acid is also a function of ionic species, for as compared to a value of about -85” in 0.2 M NaC1, 0.066 M CaClz raises [(Y]WN to about -65” while 2 M glycine lowers it to below -100” (Wada and Doty, (1961). Clupein, a disordered protein chain, is likewise sensitive to ionic strength and species (Schellman, 1958e). Side chain ionization may itself contribute t o A[m’]fi: for ionic polypeptides, but it is difficult to distinguish from the accompanying conformational changes. For y-poly-D-glutamic acid, a bacterial polypeptide that cannot form an a-helix, Edelhoch and Lippoldt (1960) have observed a radical dependence of [a1400and A, upon pH as well
+
OPTICAL ROTATION AND PROTEIN CONFORMATION
447
as ionic environment. They attribute this susceptibility to the presence of an ionizable group adjacent to the polypeptide backbone, a structure not found in standard synthetic polypeptides and proteins, so that ionization is probably of lesser consequence in these substances. The oxidized A chain of insulin, for example, displays virtually no change in or A, with extremes of p H (Schellman, 1958e). However, in view of the sensitivity of disordered polypeptide chains to ions in aqueous solution, any choice of an increment in optical rotation as representing the formation of a helix should be based upon typical solvent conditions. A value for A[m’]gf of 90” appears to be a reasonable average in 0.2 M NaC1. To summarize, a direct comparison of the dispersions for a helical and randomly coiled polypeptide chain has shown that a b f of about -630 is characteristic of the isolated helix, for this parameter, which is a measure of the complexity of the dispersion, usually has negligible values for disordered chains. As has been suggested, exceptions to this rule may be explained by discrepancies between A, of the random coil and & . Another coefficient, with simple Drude dependence is also a property of the helical conformation, but its measurement is confined to individual cases in which solvent effects over the course of the helix-coil transition are at a minimum. For water-soluble ionic polypeptides a t ionic strength 0.2, a?-D is about +650. The remaining property of the helix, its rotatory contribution a t any single wavelength, is likewise masked by solvent effects upon the random coil, so that A[m’]f-” is usually a reliable measure only in aqueous solution, and here only under specified salt conditions. A t a n ionic strength of 0.2, A[m’]f-” is about 90”. Although in the interests of clarity numerical values for l# and af-” are usually written without the designation of degrees, these coefficients have the same dimensions as A[m’]F”, that is, degrees centimeters’ per decimole (see Section 11, A ) . 2. Meso-Helices
A second way of eliminating the intrinsic residue contributions from helical dispersion is to introduce an equal number of residues with D-configuration into helical polypeptides and thus cancel a:, leaving the dispersion presumably that of the meso-helix. It might be thought that this mode of cancelling a: would not be necessary if polyglycine, for which it is zero, could be made to adopt the helical conformation in solution, but the absence of a side chain in this polymer apparently predisposes it to form intermolecular hydrogen bonds either in aggregates of the p-form or to take up a hexagonal packing in which each chain is bonded to its six neighbors (Crick and Rich, 1955). In addition, there would be no preference for rightor left-handed helices in polyglycine, so that complete cancellation might result. The practical difficulty with the meso-procedure is that the sense of a
448
PETER URNEH AND PAUL DOTY
polypeptide helix is determined in large measure by the ease with which side chains can be accommodated in a helical array, so that a given configuration of residue will favor one helical sense over its opposite. Some D-residues can be embodied in a helix with its sense determined by L-residues, but, as the fraction of D increases beyond about 0.3, either sequences of D-residue form helices of opposite sense, thus cancelling the helix of single sense which we wished to isolate, or mixtures of D- and L-residue fail to form helices and lead to regions of random conformation. Both studies that employ this method, that of Doty and Lundberg (1957) and that by Downie et al. (1957), therefore use some means of extrapolating rotations obtained at fractions of D-residue tolerated by a helix of single sense to values expected for the meso-helix. The advantage of this technique compared to that involving the random coil is that all measurements are carried out in one solvent which favors the helical form. Dispersion data based on the extrapolated values do follow a Moffitt plot in which the coefficients presumably reflect the helix itself. Poly-y-benzylDL-glutamate gives a b: of -500 and an intercept, which we may equate with a F D ,of f680 (Doty and Lundberg, 1957),while bf values for PO~Y-DLleucine range from -446 to -513 (Downie et al., 1957), all values referred to A0 = 212 mp. The data obtained from extrapolation are not precise enough to permit independent determination of Xo , but Downie et al. find that if A0 is set to 200 mp, the poly-DL-leucine data yield a bf of -640, and similar alteration would bring b: for poly-y-benzyl-DL-glutamate into line with values for polymers with residues of single configuration. Hence under these conditions, either bo or Xo changes with reference to the standard polypeptides. Tinoco and Woody (1960) point out that a meso-helix is not the same as a helix with no side chains, and that D-and L-residue interactions with, say, a right-handed helix will be different and may not cancel. Downie et al. interpret their results to indicate that the contribution of D-residues to the dispersion is greater than that of L-residues in these cases. Crystals of DLpolypeptides show some untwisting of the a-helix and changes in the infra1960b), so that a comred spectrum (Bamford et aZ., 1956; Bradbury et d., bination of this effect with the difference in D- and L-interactions may account for the moderate discrepancy in the Moffitt parameters. The A[m’]k,D values obtained on these and other polymers listed in Table I1 are all somewhat lower than those given by comparison with random coils, and they do show solvent differences for the same helical polymer, yet their relative uniformity suggests that large solvent effects characteristic for the random coil have been eliminated. The agreement of A[m’]fGD with that for polyso that one may conmers in aqueous solution is good, as is the value of clude that the meso-technique essentially corroborates the comparison of helical dispersion with that of the random coil.
OPTICAL ROTATION AND PROTETN CONFORMATION
449
If the dispersion of an isolated helix is plotted from a Moffitt equation with the coefficients found by Doty and Lundberg, it shows a point of inflection and a maximum near 400 mp and becomes progressively levorotatory below 300 mp, indicating t,hat the contribution of the helical form is indeed the anomalous variety of complex dispersion. The anomalous character of the dispersion of poly-y-benzyl-L-glutamate is especially evident in m-cresol (Fig. l ) , a solvent in which Downie et al. (1957) suggest that TABLE I1 The Rotatory Contribution of the Helix* Solvent
Polypeptide
Pol y -DL-alnnine
CHC13 , 99%; DCA,
[m‘l~
Reference
$82
Downie el al. (1957)
+81
Downie et al. (1957)
Benzene
+96
Downie el al. (1957)
+75
Downie et al. (1957)
Polymer C
Dioxaue, CHCL , pyridine DMF, rn-cresol CHCI, Dioxan e Pyridine m-Cresol DMF Dioxane
4-68 1-70 67 i-65 3-58 54 +90
Downie et al. (1957) Downie et al. (1957) Downie el al. (1957) Downie et at. 11957) Downie el al. (1957) Downie et al. (1957) Doty and Lundberg
Polymer D
CHCl,
4-78
Blout el al. (1957)
1%
Po~y-DIA-ru-amino-nbutyric acid Poly-DL-leucine 1’oly-y-bensyl-i~r.glutamate I’olynier A Polymer 73
CHC13, 90%; DCA, 10%
+ +
(1957)
* Reduced mean residue rotations for helical L-polypeptides extrapolated to mesocomposition. Solvent abbreviations: CHCla , chloroform; DCA, dichlorortcetic acid; DMF, dimethyl formamide. there is little difference between D- and L-residue contributions to the helical dispersion. The intrinsic residue contribution of poly-y-benzyl-L-glutamate in m-cresol will hence be small, thus allowing the anomalous character of the helical dispersion near 400 mp to express itself. This same effect is clear in the observed dispersions for poly-y-benzyl-DL-glutamates in dioxane with progressive enantiomorphic cancellation (Doty and Lundberg, 1957). It is also seen for silk fibroin a t high concentrations of ethylene dichloride, a solvent in which this protein is largely helical; since 44 % of the residues of silk fibroin are glycine, which has no intrinsic residue rotation, the rotatory contribution of the helical form can emerge (Yang and Doty, 1957). Thus whether the observed complex dispersion of helical polypeptides is anomalous, as for poly-y-benzyl-L-glutamate in m-cresol (Fig. l), or not, as for
450
PETER URNES AND PAUL DOTY
poly-L-glutamic acid in water, seems to depend upon the nature of the side chains and solvent interactions with them.
D. The Issue of Helical Sense That the polypeptides thus far characterized contain helices of one sense only is evident both from their hydrodynamic behavior and the study of meso-helices. A change of helical sense within a molecule will cause the breakage of several intramolecular hydrogen bonds, which in turn will produce a flexible region in the chain. If breaks were at all frequent, the molecule would obviously fail to exhibit the rodlike properties that have been observed. Moreover, the respective contributions of helices of opposite sense should essentially cancel each other. Cancellation of this sort in fact appears to take place in meso-helices, in which it is now thought that helical regions of opposite sense persist. The existence of short sequences of residues with the same configuration that can form a stable helical segment is made plausible by statistical studies on the probability of copolymerizing a given sequence of identical residues (Doty and Lundberg, 1957; Wada, 1961 ) . Experimental evidence that meso-poly-y-benzyhL-glutamate actually consists of helical segments rather than randomly coiled regions is provided by the slow rate of hydrogen-deuterium exchange (Bradbury et al., 1960b), the structural interpretation of dielectric dispersion studies ( Wada, 1961) , and the analysis of curves representing progressive enantiomorphic cancellation (Downie et al., 1957). Furthermore, the initial addition of D-residues to helical L-polypeptides leads to a linear increase in rotatory power (Elliott et al., 1956; Blout et al., 1957; Downie et al., 1957), an effect that is most easily interpreted in terms of the persistence of one sense of helix and the gradual expression of its positive rotatory contribution, that is, up to a point at which D-residues begin to determine helical sense. One may therefore conclude both from hydrodynamic evidence and the effect of introducing D-residues known to change the helical sense that L-polypeptide helices are of a single sense. It is axiomatic that enantiomorphic helices will manifest diametrically opposed rotatory properties, just as amino acids of opposite configuration will mutually cancel their rotatory power. The dispersions of polyy-benzylL-glutamate and poly-y-benzyl-D-glutamate are mirror images of each other and their parameters are almost equal and opposite in sign (Blout and Karlson, 1958). The rotatory characteristics of poly-D-alanine (Elliott et al., 1958), poly-D-a-amino-n-butyric acid (Downie et al., 1957), listed in Table 111, and poly-D-glutamic acid (Stryer and Blout, 1961) are likewise antithetical to those of most L-polypeptides. Quite apart from other evidence that will be described, this finding is in itself sufficient to establish that residue configuration can be a decisive factor in determining helical sense.
OPTICAL ROTATION AND P R O T E I N CONFORMATION
451
The rotatory behavior of poly-P-benzyl-L-aspartate presents an exception to the rule that L-residues favor helices of the same sense, for it has a bo of about +600 (Blout and Karlson, 1958), as well as a far ultraviolet dispersion and changes in [ m ’ ] on ~ forming the random coil that are obviously opposite in sign to those of polyy-benzyl-L-glutamate (Karlson et al., 1960). In addition, its enantiomorph, poly-P-benzyl-D-aspartate behaves as a normal L-polypeptide (Blout and Karlson, 1958). The systematic investigations of Bradbury et aE. (1959, 1960a, b ) and Karlson et al. (1960) leave little doubt that helical poly-P-benzyl-L-aspartate has a sense opposite to that of the standard polymers and that its rotatory parameters reflect this TABLEI11 Rotatory Properties of Synthetic Polypeptides with Helices Opposite to Standard Sense* ~
~~~
Polypeptide
Solvent
Poly-D-alanine Poly-D-n-amino-n-butyric acid
Film CHClt, 90%; DCA, 10% TFA CHCla
Poly-8-benryl-L-aspartate
Poly-y-benzyl-D-glutamate
Optical rotation
[ m ‘ l ~ -19 [Wb’lD (a1646
-
+611
-18 -172
-
-
-18
DCA
[U]646
+17
-150 -28.6
[m’isrs +zgn
All ao and bo measurements are based on Xo dichloroacetic acid.
-
-
[m’lsrs -274 Im’1648
+475
-
(a1646
[m’]646
-
-
[a1646
-
bo
+I07 -168
DCA CHCla DCA m-Cresol CHCli DCA Film CHCIa
[alsrs
-
Reference
ao
Elliott et al. (1958) Downie et at. (1957) Downie et al. (1957) Blout and Karlson (1958)
-
$630 -200 +534 4-665 -250 -3560
-
-
S615
Blout and Karlson (1958) Karlson et al. (1960) Karlson et al. (1960) Bradbury et al. (1959) Bradbury et aE. (1959) Bradbury et al. (1959) Bradbury et aE. (1959) Blout and Karlson (1968)
-
-
0
Blout and Karlson (1958)
-
-
-
-
= 212 mp. Solvent
-
abbreviations: CHClr ,chloroform; DCA,
opposite sense rather than some optical aberration. X-ray and infrared measurements indicate that this polypeptide is an a-helix both in films and in solution. There is little change in rotatory dispersion and viscosity as the fraction of y-benzyl-D-glutamateresidues copolymerized with P-benzylL-aspartate residues is increased, showing that both favor the same helical sense characterized by a positive bo . The inclusion of only a small amount of y-benzyl-L-glutamate will, however, cause sharp reversal of sign in bo to values characteristic for poly-y-benzyl-L-glutamate, again without much change in viscosity. This finding suggests not only that a reversal of sense has occurred but that the glutamate helix is much more stable than that of the corresponding aspartate. The relative instability of the aspartate helix is further attested to by its greater sensitivity to temperature and molecular weight changes, rapidity of hydrogen-deuterium exchange, and
152
PETER URNES AND PAUL DOTY
susceptibility to disruption by dichloroacetic acid. All of these properties me in accord with the steric possibilities of scale models, which show that the side chain of y-benzyl-L-glutamate is easily accommodated into a righthanded a-helix of its polymer, but not a left-handed one, whereas the shorter side chains of poly-0-benzyl-L-aspartate are difficult to embody in either helix and thus should produce inherent instability. The occurrence here of a helix of nonstandard sense with L-amino acid residues thus appears to result from the unusual interaction of its side chains, a matter which could be further elucidated by study of the rotatory properties of its simpler derivutive, poly-L-aspartic acid. Which sense, then, right- or left-handed, gives rise to the standard rotlatory dispersion? Theories of optical rotation have thus far been unable to resolve the issue, for although the right-handed assumption of Moffitt et al. (1957) and of Fitts and Kirkwood (1956a, b, 1957) receives support from experimental data, the left-handed assumption of Tinoco and Woody (1960) finds agreement with other empirical properties of these same polypeptides (see Section 11,C ) . Huggins (1952) pointed out that right-handed helices of L-polypeptides should be favored because the 0-carbon of each side chain encounters steric interference with the carbonyl oxygen of the same residue in a lefbhanded helix, and despite Donohue's demurral (Donohue, 1953), arguments from model building do seem to predict correctly extremes of helical shbility, as in the comparison of y-benzyl-gl glut am ate and 0hrnzyl-~-aspartate helices. X-ray diffract,iori has provided the strongest evidence for right-haiided heliccs in the solid state, despite the fact that Arndt and Riley (1955) have argued from X-ray data on synthetic polypeptides and proteins that lefthanded helices were present, and Brown and Trotter (1956), examining the puzzling diffraction patterns of poly-L-alanine obtained by Bamford et al. (1954), came to a like surmise. Elliott and Malcolm (1956, 1958) found that if they assumed hexagonal packing for poly-L-alanine crystals with random arrangement of chain direction, then right-handed a-helices made sense of the data while left-handed helices did not. Since all optical rotatory measurements on helical poly-~-alanine,both in films (Elliott et al., 1958) and in solution (Fasman, 19615~)yield bo values that range from -425 to - 560, this interpretation of X-ray evidence implies that negative bo values signify right-handed helices. The most decisive support for this contention has proceeded from the X-ray study of c.ryst,alline sperm whale myoglobin, which, as Kendrew al., 1960, 19G1), contains 118 of its 15:) has demonstrated (Kendrew residues, that is, 77 %, in right-handed a-helices. The side chains of L-amino acids in a right-handed helix project in a direction opposite to that of carbony1 groups hydrogen-bonded into the helix, an orientation that is in fact
OPTICAL ROTATION AND PROTEIN CONFORMATION
453
discerned a t high resolution, so that this finding, together with a knowledge of t,he absolute configuration of an L-amino acid, (Trommel and Bijvoet, 1954), establishes that the a-helices in this protein crystal are right-handed. Unfortunately, myoglobin in solution does iiot readily permit, optical rotatory assessment of its conformation because the heme group itself dominates the dispersion over most of the visible spectrum. Measurements may, however, be carried out upon its transparent globin in the wavelength range usually available for polarimetry. Dispersion data upon this heme-free protein in aqueous solution yield negative bo values which, on a rationale yet to be discussed, are compatible with a partial helical content of about 50% (Doty, 1957; Schellman, 1959; see Table XII). This estimate in all probability sets a minimum for the native protein. As judged from bo , the helical content of globin-M may be increased to 80% in 2-chloroethanol (Table XIII),a value that perhaps cannot be exceeded in view of the four proline residues in the polypeptide chain. Therefore it can be argued that the native protein has a helical content intermediate between these estimates and should indeed display a negative bo . Nonetheless, rotatory dispersion measurements on the native protein itself would carry great>erconviction that a negative bo is the correlate of a right-handed helix. Rotatory dispersion measurements have therefore been recently made on t,he ultraviolet side of the visible heme absorption bands and their attendant Cotton effects, from 240 to 360 mp, a region in which the peptide bond chromophores govern the dispersion (Umes et al., 1961a). A comparison of the ultraviolet rotatory dispersion of native myoglobin with that for a standard helical polypeptide, a 5 % copolymer of L-tyrosine with Lglutamic acid a t pH 4, clearly shows that the optical rotations of both are sharply levorotatory below 260 mp, while the respective disordered forms are distinctly less levorotatory (Fig. 9). Since the dispersion for both native myoglobin and the helical polypeptide have ba values that are large and negative, these findings establish the qualitative point t,hat both molecules in solution possess helices of the same sense. As a quantitative index of partial helical content,, bo for the native protein a t first indicated a value of more than 90 %. However, the conventional treatment of these low wavelength data was brought into question by the distinct negative curvature of the Moffitt plot in this spectral region for copoly-L-tyrosyl-L-glutamic acid in the same solvent, 0.1 M phosphate buffer (Fig. 8 ) . A slope drawn for the helical dispersion of this polymer through low wavelength points alone would hence be considerably more negative than that fitting visible and near ultraviolet data. As shown in Fig. 8, the Moffitt plot for this reference polypeptide may be straightened by increasing AO from 212 to 216 mp, an alteration that changes bo from -615 to -535 (see Section 111,23). Once the myogIobin data were recast
454
PETER URNES AND PAUL DOTY
with ho = 216 mp, and bo = -F535 taken as a new scale for full helical cont,ent,, a value of about 73% helix was obtained from a bo of -390. This value receives support from the far ultraviolet spectra of myoglobin, which (
-200(
M YO G LO0 I N 8 M UREA
/,,,/
-400(
[m'l -600(
-BOO( I
i PTG A ,
,i
pH4 I -10,Oo~
I 240
I
260
I
I
200
300
J
3;
x, mP FIG.9. Ultraviolet rotatory dispersions of sperm whale myoglobin and a copolymer of 59r, L-tyrosine with L-glutamic acid (PTGA). Myoglobin in 0.1 M phosphate buffer, pH 7.0, 0 - 0 ; in 8 M urea, 0-0. PTGA in 0.1 M phosphate buffer, pH 4.0, A - A ; pH 7.0 in the same solvent, A - A. Moffitt plots for the dispersion of PTGA in this spectral range are shown in Fig. 8. (Umes et al., 1961a.) indicate a helical content of 70% or greater in terms of the characteristic hypochromism of the peptide bond at 190 mp (Rosenheck and Doty, 1961; Table XV). Beychok and Blout (1961), who have also measured the ultraviolet rotatory dispersion of myoglobin, judge the helical content to be 7 5 4 0 % from b, with A 0 set t o 212 mp. The solvent used in this study was water with zero ionic strength; this condition can perhaps account for the difference in A 0 from the work described above, for 1 0 may in this wave-
OPTICAL ROTATION AND PROTEIN CONFORMATION
455
length range be dependent upon environment. They have, moreover, carried the dispersion beyond the minimum at 233 mp (Fig. 4) and hence derive a second estimate of helical content, again 75-80 %, from the optical rotation at this wavelength. Their finding that myoglobin, like helical polypeptides, exhibits a minimum of large amplitude near 233 mp further strengthens the identity of helical sense in these molecules. The quantitative agreement of rotatory dispersion with helical content in the crystal is good evidence that the secondary structure of this protein, consisting of right-handed a-helices, persists in solution. Thus, save for the unlikely contingency that myoglobin opens and then refolds into helices of opposite sense upon entering solution, the correlation of right-handed helices with a negative bo and its associated parameters is virtually certain.
E. Optical Rotatory Dispersion of Polypeptide Films The synthetic polypeptide most completely studied in the solid state by optical rotation is poly-D-alanine, which Elliott el al. (1957b, 1958) treat as though it were an L-polypeptide with opposite helical sense in order to make straightforward comparison of the results with other studies. The bo for the pure L-polymer is -475, and for two helical DL-copolymers, -500 and -505; extrapolated values for the meso-polypeptide lead to a bo of -560. These compare favorably with Fasman’s result of about -425 in 30% dichloroacetic acid in chloroform, a solvent in which the conformation is helical (Fasman, 1961a). The [m’]~values were found to be less than those obtained for solution, a result probably attributable to environmental changes, yet A[m’]?-’-”of meso-poly-m-alanine is +70° at 578 mp, which is in reasonable agreement with the value of +SO0 at 589 mp for its counterpart in solution (Downie et al., 1957). Infrared spectra of these same films show bands associated with a-helical poly-L-alanine, the presence of which was established by X-ray methods. The rotatory attributes of this helical polypeptide in solution have therefore received conformational assignment both in terms of the fundamental criterion of X-ray diffraction and through the sequential application of auxiliary criteria, those of infrared spectra and now optical rotatory dispersion, that can bridge the crystal and solution. Few further correlations of optical rotation and solid state properties have been made, but it is evident t,hat environmental effects can produce large changes in bo as well as in [m’]~Films of poly-L-glutamic acid in the form of sodium and potassium salts, which give no sign of @-structuresand hence are presumably in the random coil, yield a bo value of about -70 (Elliott et al., 1958), and helical poly-y-benzyl-L-glutamate in the solid state has a bo of -800 (Elliott et al., 1961), both of which values are of the expected order. Yet poly-P-benzyl-L-aspartate films cast from chloroform have a bo of -3560 and [ m ’ j of ~ +290°, a striking change from chloroform solution
.
456
PMTEEl LJHNMS AND PAUL DOTY
in which they are $600 and -270") respectively (Bradbury et al., 1959). The same side chain chromophores exist in poly-p-benzyl-L-aspartate as in poly-y-benzyl-L-glutamate, but since they are closer to the helical backbone in the latter polymer, they might assume periodic order in the solid state and therefore contribute strongly to rotatory dispersion. Therefore it may be more than a coincidence that films of poly-L-tyrosine cast from dimethyl formamide, in which infrared and X-ray evidence suggest that t,he polypeptide is a-helical, have a bo of -4000 (Downie et al., 1959)) for both polymers have benzenoid chromophores close to the main polypeptide chain. In dimethyl formamide solution, bo values of +400 (Downie et al., 1950) and +550 (Coombes et al., 1960) have been obtained for poly-Ltyrosine, findings which will be discussed in the next section, so that large rotatory differences between solid and solution exist for this polymer as well.
F. Synthetic Polypeptides with Unusual Rotatory Attributes IVot all synthetic polypeptides exhibit the standard correlation of rotatory properties with the helical and disordered conformations. The exceptions present a twofold challenge, for they imply that the established correspondence between rotatory dispersion and the a-helix may not be unique and at the same time introduce complications involving amino acids that must, be taken into account in the study of protein structure. Although the investigation of some of these polypeptides is as yet fragmentary, enough information exists to suggest that they can indeed be seen as exceptions, either because of strong chromophoric contributions of side chains, as in the case of poly-L-tyrosine, or because they form structures other than the a-helix, as does poly-L-proline. It is important to emphasize at the outset that amino acids with exceptional chromophoric properties occur to such a small extent in proteins that their intrinsic properties do not obscure the essential features of the rotatory dispersion. Proline and hydroxyproline introduce a stJeric discontinuity between helical segments such that they can influence the conformation of proteins wherever they occur. However, it is only in the collagen class of proteins, which consist preponderantly of these two species of amino acid with glycine, that the conformational features of poly-L-proline become manifest in the unique triple-stranded collagen helix. Of polypeptides with unusual chromophoric attributes, poly-L-tyrosine is the most, thoroughly studied. X-ray and infrared evidence demonstrate that this polypeptide is a-helical in films cast from dimethyl formamide (Downie et al., 1959), and hydrodynamic studies have established that it exists as a helix in solution as well (Coombes et al., 1960). The helical form, which is stable in both dimethyl formamide and aqueous solution of pH 10.85, may be transformed into the random coil either by increasing the
OPTICAL ROTATION AND PROTETN CONFORMATION
457
polarity of the solvent with dichloroacetic acid or by ionization of the side chains. The existence of the random coil was demonstrated not only by the lack of positive birefringence but also by the sensitivity of viscosity and sedimentation to ionic strength, a characteristic of flexible polyelectrolytes. Its rotatory properties, listed in Table IV, are, however, clearly at variance with the usual pattern, for although A[a]EiDis in a levorotatory direction typical for disruption of the helix in standard polypeptides, the dispersion of helical poly-L-tyrosine is simple with X, = 237 mp, a parameter which increases progressively through the helix-coil transition to a value of 330 mp. The change in bo is also in the customary direction, but not only is it comparatively small, the values for both conformations are in addition large and positive. The behavior of poly-0-carbobenzoxy-L-tyrosine is apparently similar (Katchalski, 1958). That the poly-L-tyrosine helix is of standard sense has recently been ascertained by Fasman (1961b), who found that a series of copolymers containing varying proportions of L-glutamic acid and 1,-tyrosine displayed a linear change in bo between -560 for poly-L-glutamic acid to +494 for poly-L-tyrosine in a solvent, dimethyl sulfoxide, in which all copolymers were helical. Had there been a change in helical sense at some intermediate composition, bo would be expected to show a sudden shift such as that found in copolymers of y-benzyl-L-glutamate and @-benxyl-L-aspartate(Karlson et al., 1960; Bradbury et al., 1960b). This result further suggests that the contribution of tyrosine to bo arises from local interactions of the side-chain chromophore with the peptide bond, so that it is characteristic of an individual residue in the helix. This interpretation is supported by the unusual rotatory properties of both the random coil and the free amino acid. The distinct properties of tyrosine were revealed in the investigations of Schellman (Schellman and Schellman, 1956; Schellman, 1960), who found that the dispersions of tyrosine, tryptophan, and phenylalanine, unlike those typified by alanine, are complex and appear to implicate the aromatic absorption bands in the region 260-290 mp. Thus the uncommon attributes of poly-L-tyrosine may be tentatively referred to interactions inherent in each residue that are superimposed upon the ordinary dispersion of the helix. As shown in a recent study (Urnes et al., 1961b), a copolymer of Lglutamic acid with 5 % L-tyrosine has completely normal dispersion characteristics in both the helical and random coil conformations and displays no measurable Cotton effects at the tyrosyl absorption band (Fig. I)), even though these might have been anticipated from the properties of the free amino acid. Although it is possible that tyrosyl-tyrosyl interactions contribute to the helical dispersion of poly-L-tyrosine, it is quite uiilikely that they affect helical dispersion in proteins, in which tyrosine is greatly diluted by other residues. The rotatory behavior of poly-L-tryptophan in dimethyl formamide (Sela
458
PETER URNES AND PAUL DOTY
TABLEIV Synthetic Polypeptides with Unusual Rotatory Properties*
--
Solvent
Polypeptide
Optical rotation
hc
bo
Reference
235 330
+550 $470
Coombes et al. (1960) Coomhes et al. (1960)
237
+540
Coornbes et al. (1960)
330
$450
Coornbes et al. (1960)
-
-4000 +494 $263
Downie et al. (1959) Fasrnan (1961b) Fasman (196lb)
-
+270 4-40
Katchaleki (1958) Katchalski (1958)
235
4-410
-
Sela et al. (1961) Sela et al. (1961)
NL
NL
Norland et al. (1961)
206
4-330
Norland et al. (1961)
285 179
$75
-
Norland el al. (1961) Norland et al. (1961)
217
-
Norland et al. (1961)
224
__
Norland et al. (1961)
-
$250
md
ao
Poly-L-tyrosine
Poly-L-tyrosine, sodium salt Poly-0-carbohenzoxy-Ltyroaine Poly-L-tryptophan
Poly-1-henzyl-Lhistidine
Pol y-I-histidine
Poly-0-acetyl-L-serine
Poly-L-serine
Poly-L-proline, form I Poly-L-proline, form
DMF DMF, 20%; DCA, 80% €120, 0.15 M NaCl, p H 10.85 HzO, 0.15 M NaCI. pH 12.27 Film Dimethyl sulfoxidc Dirnethyl sulfoxide
-
-
DMF TFA DMF DMF, 40%; DCA, 60% CHCls; DCA per residue, 0.2 CHC1,; DCA per residue, 1.0 DCA Hz0, 0.2 &f NaC1. p H 2.4 HzO, 0.2 M NaC1, pH 5 85 HzO, 0.2 M NaC1, pH 6.00 2-Chloroethanol, W%; DCA, 10% DCA EDC, 75%; DCA, 25% DCA
-
-
4-300
214
0
120
0
HzO, 10 M LiBr
290
0
HsO, 8 M urea
250
0
Acetic acid
NL
Acetic acid
206
Formic acid, 90%
-
-
Katchalski (1958) Fasman and Blout (1960) Faaman and Rlout (1960) Fasruan and Blout (1960) Fasrnan and Blout (1980) Blout and Fasman (1957) Blout and Fasnian (1967) Kurtz et al. (1958)
Formic acid, 90%
-
-
Kurtz
Ha0
-
-
Kurtz et al. (1958)
I1 Poly-0-acetyl-L-hydroxyproline, form
I Poly-0-acetyl-L-hydroxyproline. form
I1 Poly-L-hydrox yproline
Katchalski (1958)
-
N 1, 0
et
al. (1958)
All measurements of ao nnd bo are based on X o 212 rnp. Solvent abbreviations: CHCls , chloroform; DCA, dichloroacetic acid; DMF, dimethyl forrnamide; EDC, ethylene dichloride. NL: nonlinear plot.
OPTICAL ROTATION AND PROTEIN CONFORMATION
459
et uZ., 1961) is strikingly similar to that of poly-L-tyrosine and may likewise be referrable in part to the unusual properties of the constituent amino acid, but the change in [m']646on going to dichloroacetic acid is in the opposite direction, increasing by 165'. In terms of optical rotation, dichloroacetic acid appears to bring about a conformational change, but unfortunately the hydrodynamic evidence for the existence of helices in dimethyl fonnamide is not definitive. Since the indole side chain appears to react with dichloroacetic acid, this complication should be taken into account in assessing rotatory behavior in this solvent. An analysis of poly-l-benzyl-L-histidineand poly-L-histidine by infrared spectroscopy and optical rotation has led Norland et al. (1961) to propose that these polypeptides form helices opposite to the standard sense, that is, left-handed. The infrared, absorption frequencies of poly-l-benzyl-L-histidine in chloroform suggest that it is an un-ionized random coil, and since in dichloroacetic acid the side chain becomes ionized, one would expect the polymer to be disordered in this solvent as well. Yet a t an equimolar ratio of dichloroacetic acid to 1-benzyl-L-histidine residue, this polypeptide forms a helix as judged from the infrared dichroism of films. That this helix exists in solution is indicated by the marked decrease in & [, , from +25" to -208", as acid is added to a chloroform solution and the dichloroacetate salt is formed, followed by a rapid rise toward more positive values once equimolar ratio is passed, and it finally reaches +28" a t 100% dichloroacetic acid. The dichloroacetate salt would appear to have the same helical sense as poly-@-benzyl-L-aspartate, which has been demonstrated to be opposite to standard helices (Section 111, D).The visible rotations of both polymers decrease significantly on forming helices, and both have infrared absorption frequencies that differ characteristically from standard helical polypeptides. Furthermore, the dispersion of the trifluoroacetate salt, like that of helical poly-0-benzyl-L-aspartate, becomes 'progressively more positive below 250 mp. Although it has been less thoroughly investigated, poly-L-histidine also appears to form a helix that is left-handed. Soluble in water, it is ionized at low pH and presumably disordered. As p H 6 is, approached, [a1646 decreases from about -65" to a sharp minimum at -210" and then rises a t once to -67", thus displaying the same sequence as its parent molecule in chloroform-dichloroacetic acid mixtures. With the exception of un-ionized poly-l-benzyl-L-histidine in chloroform solution, which gives a positive dispersion that fits neither the simple Drude nor the Moffitt equation and is hence similar to form I of poly-L-proline (Table IV), all conformations of these two polypeptides display simple dispersion. The dispersions of un-ionized and completely ionized poly-l-benzyl-L-histidine are similar to those of poly-L-tyrosine and poly-L-tryptophan in becoming progressively more positive as the wavelength is decreased. Although free
460
PETER UIENES AND PAUL DOTY
histidine does not share the unusual rotatory properties of aromatic amino acids, i t has absorpt,ion bands further in the ultraviolet which could endow it with strong side chain effects. Nearly all the optical rotation data in the foregoing exceptional cases can be described by simple dispersion with a variety of A, values. Treatment of the same data by the Moffitt equation therefore introduces pseudocomplexity (see Section 111,C , I ) , although it does bring out a further similarity among the dispersions of poly-L-tyrosine, poly-L-tryptophan, and poly-l-benzylL-histidine in their large, positive bo values. The significance of this trend may become apparent by further study of copolymers of these amino acids with standard residues for which the Moffitt parameters have established meaning. The ascription of these exceptional properties to side-chain chromophores close to the main chain, as represented by Blout's classification of these polypeptides in a separate group, is a good working generalieation (Blout, 1960), one which is reinforced by Schellman's very similar assignment of rotatory differences among individual amino acids (Schellman, 1960). Poly-O-acetyl-L-serine (Fasman and Blout, 1960) exists as a random coil in dichloroacetic acid, giving simple dispersion charact'erized by A, = 120 mp, bo = 0, and [?)2']646 = +as0, but upon mixture with less polar solvents takes up t.he p-conformation instead of an a-helix, as shown by infrared measurements. The large increases in [m']646to + l l 9 " and A, to 210 mp wit,hout a change in 6 0 presumably represent the formation of the p-form. This matter will be furhher discussed in the section on the rotatory properties of this conformation. The failure of poly-0-anetyl-L-serine tJo form a helix is conceivably tl result of skric interference by t,he acetyl group. It appears, however, that its simpler derivstive, poly-L-serine, is likewise incapable of assuming a helical conformation. The perplexing result that poly1,-serine formed only a random coil in aqueous solution, as displayed by its infrared spectra and rapid hydrogen-deuterium exchange ( li'asman and Blout, 1960), might, be explaiiied by a current report that this polymer can be partially racemized during deacetylation with sodium methoxide (Katchulski and Steinberg, 1961). If this had occurred, t.he u-residues formed might prevent helix formation, and their presence might account as well for the lack of systematic variation of A, for the simple rotatory dispersion of poly-L-serine with bo . Yet since poly-L-serine prepared without racemization gives only t,he @-formin the solid state (Fasman and Blout, 1961), the initial conclusion t>hatt,his polypcptide does not form a helix in solution is probably correct. This finding does not, however, entail that a single serine residue polymerized with ot'her amino acids, as in a protein, will prevent helix formation in its vicinity, for the failure of the polypeptide to form a helix may result from the mutual interference of like side chains.
OPTICAL ROTATION AND PROTEIN CONFORMATION
46 I
Poly-L-proline and the protein collagen for which it and polyglycine serve as models are the subject of a critical review in this volume by Harrington and von Hippel, so that only the principal facts of its rotatory behavior and conformation will be mentioned here. By virtue of its pyrrolidine ring, proline embodied in a polypeptide chain has no imino hydrogen to donate for an intramolecular bond and is further limited in its conformational adaptability by the lack of free rotation about the nitrogen-a-carbon bond. Polypeptides of proline and hydroxyproline hence cannot adopt the a-helical conformation. Poly-L-proline nonetheless exists in two forms, each with distinctive rotatory properties that have played a central role in identifying the respective conformations as helical structures and elucidating the chemical basis of their interconversion. Form I, characterized by an optical rotation [m']648= +36" and a dispersion that fits neither the simple Drude nor the Moffitt equation, undergoes mutarotation in acetic acid to form 11, recognized by [~,']6~ of -578" and simple dispersion with A, = 206 mp, a process that is reversible in the appropriate mixture of less polar solvents (Kurtz et al., 1956; Blout and Fasman, 1957). The mutarotation can be followed by A, as well as the optical rotation, for the dispersion becomes simple shortly after form I is dissolved in acetic acid, with a A, of about 100 mp (Steinberg et al., 1960a, b ) . The mutarotation has been ascribed to a cis-trans-isomerizatioii about the peptide bond (Kurtz et al., 1956), the kinetics and catalysis of which have been thoroughly investigated (Steinberg et al., 19601, b ; Dowriie and Randall, 1959). The X-ray analysis of form I1 by Cowaii arid McGtLvin (1955) revealed an extended helix with lefbhanded twist and imide groups in the trans-position in the backbone. Proceeding from hydrodynamic evidence indicating the presence of rigid rods, Harrington and Sela (1958) used the polarizability theory of Fitts and Kirkwood ( 1956a) with these crystallographic dimensions to compute the helical contribution to the specific rotation of form I1 and were able to conclude that its structure in the crystal persists in solution. On the basis of a more compact helical model with a right-handed twist and imide bonds in the &-position, they were able to predict the relatively positive rotat,ion of form I , which also behaves in solution as a rigid rod. I t is important to note that neither form I nor form I1 is likely to occur in proteins with small proportions of proline and hydroxyproline, but, as previously mentioned, these residues will interfere with the formation of a-helices. With the use of models it has been shown that proline can be incorporated a t the amino end of a right-handed a-helix through hydrogen bonds to its carbonyl oxygen, and that the helix, although weakened, can continue beyond it (Low and Edsall, 1956). It cannot, however, be aceommodated in a left-handed helix without drastic alteration of the direction of the following helical segment. If proline has the &-configuration, then in
462
PETER URNES AND PAUL DOTY
a right-handed helix it permits a sharp corner to be made and the next helical segment to run in the reverse direction (Edsall, 1954). The X-ray investigation of sperm whale myoglobin a t high resolution (Kendrew et al., 1961 ) demonstrates that these stereochemical features of proline in fact influence protein conformation, for all four prolines in this molecule, which are uniformly situated at amino ends of right-handed a-helical segments, introduce discontinuities in the secondary structure. Indeed, one of these corners between helical segments is neatly turned a t the proline junction wit,hout the appearance of a disordered region. Quite aside from its steric influence, proline can affect the mean residue rotation of a protein by its comparatively large levorotation (Schellman, 1960). Since poly-L-proline does not exist as a random coil, the intrinsic residue rotation of an interior residue cannot be obtained as directly as for other polymers. A variety of independent estimates do, however, point t,o a value of about -2250" for the specific rotation a t 589 mp (Harrington and Sela, 1958). The viscosity of form I1 of poly-L-proline is found to decrease in aqueous solutions of saturated lithium bromide and approach that of a random coil, with an increase in the specific rotation to -250". Furthermore, the limiting rotation of proline in small peptides and copolymers of proline and glycine is in reasonably good agreement with this figure, as are values estimated both from the rotation at the transition point during the mutarotation and the theoretical helical contribution superimposed on the proline rcsidues in forms I and 11.
G. The Mofitt Equation for Mixtures of Helices and Random Coils
As we have seen, the helical and disordered forms of standard synthetic polypeptides display characteristically different properties of optical rotatory dispersion. Whereas the dispersion of the random coil is simple, that of a polypeptide known to be helical requires a complex expression for its description. Given this distinct difference, it therefore becomes possible to use rotatory dispersion as an analytical method to decide whether a po1ypept)ide of uncertain conformation is a perfect helix or a complete coil. Compositional heterogeneity of the sort found in proteins, in which twenty-odd amino acids in unique sequence replace the one or two studied in a given synthetic polypeptide, may of course complicate the assignment, but if residues with unusual rotatory attributes occur in the small proportions typical of proteins, the characteristic difference between helical and random dispersion will not be obscured. Yet proteins differ from synthetic polypeptides in another fundamental respect, for they can contain within a single molecule two or more stable conformations of the polypeptide chain and hence display a het,erogeneity in secondary structure, one that may have its ultimate ground in a particular sequence of residues. The essential challenge
OPTICAL ROTATION AND PROTEIN CONFORMATION
463
that an unknown protein structure thus presents for rotatory dispersion involves its capacity to sort out mixtures of conformations that it can discriminate singly in analogous but simpler molecules. Fortunately, for one pair of possible conformations, helices of standard sense and disordered chains, this capacity of the technique has been tested with several synthetic polypeptides within individual molecules of which independent evidence indicates the simultaneous presence of helical and random regions. Before looking directly a t these results, let us first set out a formal combination of helical and random dispersion on the assumption that helical and disordered structures contribute to the total rotatory dispersion in a strictly proportional manner. By this means, the way in which the dispersion parameters may be expected to vary with helical content will become clear. The optical rotation of a mixture of random regions and helical segments within a single molecule can be expressed as the sum of the mean residue rotations for a random coil and a helix, [m’]:and [m’];,respectively, each weighted according to that fraction, fD or j H , of the total number of residues which is embodied in the respective conformation. The net rotation of the mixture is thus
+
[m’lx = jD[m’]: jH[m’If
(30)
The assumption underlying this equation, that the mean residue rotations obtained from measurements on long random coils and helical structures will adequately represent shorter lengths of chain expected in mixtures, must, however, be examined in the light of end effects. Because the rotatory power of a residue is dependent upon its environment, its proximity to the end of a structure will endow it with a rotation that is not representative of residues a t the interior, each of which may be considered to exist in a n equivalent environment. Residues a t the ends of random coils and helical segments therefore will not contribute to the net rotation in a manner proportional to the length of the structure. 1. End Ejects in Random Coils and Helices Investigations on small peptides and randomly coiled polypeptides, discussed by Schellman and Schellman (1958) and by Doty and Geiduschek (1953) , show that the more positive rotations characteristic of residues in terminal positions decrease rapidly with the first increments in chain length and then approach values observed with long random coils more slowly. As judged from poly-L-lysine (Erlanger and Brand, 1951; Becker and Stahmann, 1952), in which the positive molar rotation of +11” for the dimer drops to -90” for a chain of about twenty residues, and then to -101” once a chain of more than a hundred residues has been reached, end effects are largely masked in a random coil of about twenty residues. Goodman’s
464
PETER TTRNER AND PATJL DOTY
work (Goodman et al., 1961) indicates that the limiting rotation may be reached with even shorter chains, for the poly-7-methyl-L-glutamate chain of eleven residues displays a specific rotation in dichloroacetic acid, -36", similar to the polymer of about one hundred and forty residues in the same solvent, -31". It appears that the dispersion properties of random coils of poly-ymethyl-L-glutamate in dichloroacetic acid, A, and bo , do not change with molecular weight, so that end effects in random coils may be expressed primarily in the rotatory power, [ m ' ] ~. Terminal effects of this latter sort will not affect disordered regions between helical segments, so that for sufficiently long chains, the use of [m']:is reasonable. It is more difficult to justify the use of [m']:for helices, however, both for the reason that end effects can be expected to involve a greater number of residues in a helical structure than in a random coil and because experimentation has as yet given no clear indication as to their magnitude. The features peculiar to helical structures that will enhance the iiifluence of end effects have been clearly elaborated by Schellman and Schellman (1958). The rigidity of a helix may provide end effects with a greater range than is possible in a flexible coil, and, more importantly, because of the relatively great length of polypeptide chain wound into a helix, more residues are near ends of this ordered structure than would be the case for an extended chain. At each end of a helical segment three residues will not he hydrogenbonded, so that each constitutes a terminal residue. Since the characteristics of helical dispersion arise from the mutual interaction of the peptide bonds, terminal residues that can interact only to one side will contribute less to helical dispersion than those in the interior. If therefore, the mean residue rotation taken as representative of helical dispersion is measured upon a very long helix in which end effects are negligible, its use in connection with shorter helices will yield an underestimate of the number of residues actually incorporated in the structure. A further consideration leading to an underestimate in very short helices is that the number of peptide bonds in a helix is one less than the number of residues, so that the mean contribution of t,he actual number of active chromophores will be somewhat diminished when placed on a residue basis. The extent to which this underestimation is a drawback has as yet proved unamenable to systematic experimental assessment. The mean residue contribution in a very long helix is in fact given by the standard, high molecular weight polypeptides, but to obtain a comparable value for short helices, one must know the number of residues in the helical conformation with certainty. One manner of proceeding is to interpret the rotatory properties of short chains in helix-promoting solvents on the assumption that the chains will be completely helical once a minimum length is reached. The minimum length required for helix formation appears to be six residues by infrared
OPTICAL ROTATION AND PHOTEIN CONB’ORMATION
465
analysis (Idelson and Blout, 1957) and about eight to twelve judging from the kinetics of polymerization (Lundberg and Doty, 1957); this number will, of course, vary with solvent. Goodman (Goodman and Schmitt, 1959; Goodman et al., 1960, 1961) has recently studied the rotatory properties of oligomeric peptides of y-methyl-L-glutamate in helix-promoting solvents and indeed has evidence that these begin to change in the direction appropriate for helical structures at about the pentamer. With further increments in the chain length, bo decreases and A, increases, displaying rapid changes when nine to eleven residues are present. The nonapeptide is the first chain which, if completely helical, contains a residue that is hydrogen-bonded at both its carbonyl and amido groups and hence qualifies as a nonterminal residue. The parameter bo does not approach large negative values characteristic for long helices of this composition until twenty-two residues are in the chain. The same result has been obtained by Mitchell et al. (1957) for poly-r-benayl-L-glutamate, which, a t a chain length of fourteen residues in dimethyl formamide and dioxane, gives a bo of about half the value characteristic of much higher molecular weight. On the assumption, then, that these polypeptides of increasing molecular weight are completely helical, the evidence indicates that rotatory contributions characteristic of helices begin to appear only with interior residues and that end effects become negligible only with chain lengths of more than twenty residues. Optical rotatory dispersion cannot, however, be used both to establish that polypeptide chains are completely helical and to assess the influence of end effects upon that dispersion. I t is therefore possible that the short chains which have been studied either represent an equilibrium between helical and randomly coiled forms or consist of helical cores with random tails. Terminal hydrogen bonds in a helix may be more labile than those a t the interior, as suggested by the hydrogen-deuterium exchange studies of Bradbury et al. (1960b) on poly-y-benzyl-L-glutamate, a finding that is also compatible with the existence of small random regions at the ends of helices. A further complication in the foregoing studies is that short chains in a helix will be subject to terminal influences similar to those found in random coils, an effect for which Goodman et al. (1961) are now able to make explicit corrections. If samples of low molecular weight in helix-promoting solvents in fact have some random content, then the above estimate of end effects in the helix will be higher than is warranted, so that the influence of terminal residues may be negligible for helices containing less than twenty residues. Two theoretical estimates of end effects in fact, suggest that they are negligible for segments smaller than this. Zimm et al. (1959), using the polarizability theory of Fitts and Kirkwood ( 1956a), found that most of the rotation characteristic of the helix should be present with one completc
466
PETER URNES AND PAUL DOTY
turn. Tinoco et al. (1961) have predicted from an exciton treatment that about 90 % of the hypochromism characteristic of the helical conformation develops with a helical assembly of eight peptide bonds, so that this effect may have its counterpart in the rotatory manifestations of the helix as well. From an empirical standpoint, it is significant for this problem that rotatory dispersion measurements on sperm whale myoglobin are in good agreement with helical content in the crystal (Beychok and Blout, 1961; Urnes et al., 1961a; see Section 111,D).Five of the eight helical segments in the crystalline protein contain from fifteen to twenty-four residues, while three of them have only seven to nine residues (Kendrew et al., 1961). If end effects were important in helices as short as this, then rotatory dispersion should yield an appreciable underestimate of the helical content of this globular protein in solution. As in other cases, rotatory dispersion cannot be used here to assess both the helical content and end effects, but since the helical content of myoglobin in solution is unlikely to be greater than it is in the crystal, 77 %, then the estimate of 70 to 80 % helix by rotatory dispersion is compatible both with the persistence of these helical segments in solution and the relative unimportance of end effects. With the recognition, then, that the use of [m’]: obtained from long helices may yet entail an underestimate of the number of residues in a short helix, Eq. (30) will be adopted as a basis for the analysis of mixtures of helices and random coils.
2, The Relation of the Mofitt Parameters to Partial Helical Content
If the expressions in Eqs. (23) and (24) are substituted into Eq. (30), an equation for the optical rotation of the mixture is obt,ained in terms of the dispersions characteristic for each conformation.
This can be reduced to a two-term expression if A, is assumed to be equal to Xo and further simplified if the variable fD is replaced by its equivalent, (1 - f a ) . (32)
The notation in this formula may be clarified by substituting a: for aD, as is discussed in Section 111, C, 1. With the aid of Eq. (29), the term (a; - a D )t,hus becomes a t F D .We thereby obtain a n expression equivalent to Eq. (32),
OPTICAL ROTATION A N D PROTEIN CONFORMATION
467
This equation for the mixture is thus of Moffitt form and is characterized by coefficients each of which is a linear function of fH and the constants appropriate for a complete coil and a perfect helix. That [m‘]~ is also a linear function of f H and constants pertaining to the two forms is shown by recasting Eq. (30) in terms of fa alone. [m’]h =
[m’]:
+
fH(
[m’lf
-
[m’]:)
(34)
Since Eq. (33) contains only two terms, the coefficient of each can be experimentally determined for a given set of data from the intercept and slope of a Moffitt plot. These coefficientswill, in this analysis, be designated a:bs and bibs, respectively, to distinguish their experimental origin clearly from the interpretive constants with which they may be equated, as follows: a:bs = a f f f H $ - D (35)
b;lbs = fJ# (36) The latter expression shows that the magnitude of bibs is directly proportional to helical content, as suggested both by Cohen and Szent-Gyorgyi (1957), who pointed out the essential requirement that A, of a disordered form must equal if bibs is to vanish with complete loss of helical content, and by Yang and Doty (1957). The merit of having bibs a direct reflection of helical content, together with the issues surrounding the approximation that A, equals Xo , has already been discussed in Section 111, 6, 1. It must, however, be emphasized that the alternative derivation indicated there, one which avoids this approximation and instead casts the dispersion of the disordered chain in Moffitt form by Eq. (28), may equally well be used for mixtures with the result that bibs loses its unique status and, in analogy to the intercept aibain Eq. (35),requires explicit measurement of b f and bf-D for its conformationalinterpretation. Although a derivation based on the equality of X, and Xo will be retained here for setting up a standard pattern of analysis applicable to proteins of unknown conformation, this alternative treatment should be used for precise studies of single polypeptides with clearly defined reference conformations and solvent conditions. The manner in which fx is related through [m’]~ , u ~ ~ and ’ , bibs to the constants for the pure forms can also be seen by solving Eqs. (34), (35), and (36) for f H . The result is
which may equally well be put in terms of the increments involved,
408
PETER TJRNES AND PAUL DOTY
Equation (38) shows that all three parameters may be expected to vary in a systematic manner with helical content when compared to a common reference state, the disordered polypeptide chain. Although these three measures of helical content are, in this formal analysis, dependent upon each other, their experimental interdependence is not complete, so that an empirical test for their mutual agreement can be carried out. For any given set of dispersion data cast in Moffitt form, it is true that the slope and intercept will depend upon individual values of [ d ] X a t the various wavelengths. But there is no necessity that the dispersion characteristics of another set of data for the same polypeptide under different conditions will, when compared with the first dispersion, display the covariance implied by Eq. (38), for in any comparison of different dispersions, two of the three increments will always be independent of one another. For example, the difference in slope may not correspond to the difference in [ W L ’ ] ~ at some single wavelength, although if this is the case, the difference in intercept will necessarily fail to agree with [ W L ’ ] ~ for the reason that it will be determined if the other t,wo values are already specified. With the understanding that only two of the increments can furnish independent information, all three may nonetheless be measured for the sake of complete characterization. If, therefore, these three measures in fact follow one another over the course of a helix-coil transition, their concurrence will provide evidence that Eq. (33) is capable of describing mixtures of these two conformations and can yield an estimate of the helical content. 3 . Partial Helical Content in Synthetic Polypeptides
A direct test for the adequacy of Eq. (33) is afforded by the rotatory benavior of poly-L-lysine over its helix-coil transition, a process shown by several lines of evidence to proceed through intermediate stages of partial helical contcnt within the same molecule (Applequist and Doty, 1961). As the side chains of poly-L-lysine lose their charge a t high pH, the helical conformation becomes stable. The first indication that this transformation did not, involve an equilibrium between perfect helices and complete coils was the absence of a sharp thermal effect, upon the transition and a correspondingly low heat of formation for the helix. Furthermore, if the molecular population were partitioned into two pure forms by an all-or-none process, then hydrodynamic methods should detect the presence of rodlike particles throughout the transition. Yet, instead of displaying a linear increase with pH, the viscosity reaches a minimum at the mid-point of the change in optical rotation, a finding compatible with the compact structure of a partially helical chain as compared to either its random coil or helical rod. Sedimentation shows only one component, a t all pH’s, thus indicating a homogeneous
OPTICAL ROTATION AND PEOTEIN CONFORMATION
469
population, and it has a maximum a t the mid-point, again to be expected for compact particles. That rodlike particles exist only a t the helical end of the transition is shown by the rapid drop in flow birefringence early in the course of the disrupting the helix by lowering the pH.
PH FIG. 10. Partial helical contcnt,fH , of poly-1,-lysine in water a t stages i n its helixcoil transition as determined by increments in three parameters of optical rotatory dispersion [Eq (38)]. A [ m ' ] ~ ~ ~ , b , " ' D / A [0, n; '(a:b'-D/uf-D, ]~~D, 0;bo"b"/bf,A. (Applequist and Doty, 1961.)
Rotatory dispersion measurements made a t numerous pH's throughout this transition all fit Moffitt plot,s. Most importantly, the increments of their parameters and optical rotations a t 589 mp as obtained from Ey. (38) all follow each other to within 3 % in tracing out the sigmoidal course characteristic of helical formation by a cooperative process, as is clearly shown by Fig. 10. Although no independent estimate of helical content was
470
PETER URNES AND PAUL DOTY
made, it would be expected on other grounds (see Section 111,H ) to change with pH in the manner described by the optical properties. Thus, in a case for which there is well-documented evidence that each polypeptide molecule contains a mixture of helical segments and disordered regions, optical rotatory dispersion cast in Moffitt form on the hypothesis that such a mixture exists gives self-consistent results. It moreover offers in the Moffitk parameters and the optical rotation at any given wavelength three separate indices, any two of which are independent, for measuring partial helical content. The conditions under which this pattern of analysis can be effective are clearly displayed in the case of poly-L-lysine. Firstly, the dispersion parameters of the two reference conformations, the helix and the random coil, must be known in order to obtain the scales on which the dispersion properties of an unknown mixture are placed, as shown in Eq. (38). Secondly, in order that Eq. (31) can be reduced to a tractable number of coefficients, and a t the same time have boObs become a direct measure of helical content, A, of the random coil must be about equal to A0 , a requirement met by poly-L-lysine for which A, in the random coil is 210 mp. A third condition is that little solvent change take place as one reference conformation is transformed into the other; if it did, then the scales for the change in rotatory power, A[m’]:-D,and the intercept, a f - Dwill , no longer be proportional simply to the helical content. The advantage of using the change in which is relatively insensitive to solvent effects as compared to these other measures, is thus apparent. The transition of poly-L-lysine does meet this stipulation, and the concurrence of [m’]689and aEbswith bibs is evidence that solvent effects are minimal. This polypeptide is, moreover, compositionally homogeneous, so that all helical and all disordered regions may be taken as equivalent to their respective reference conformations in their rotatory attributes. The assumption that other conformations, such as helices of opposite sense and p-forms, are absent during the transition is here plausible, for there is a gradual change from one established reference conformation to another. Although rotatory dispersion gives self-consistent results in the case of poly-L-lysine, it would clearly be of value to compare this method with an independent estimate of helical content. This type of correlation has been carried out in the investigation of a series of copolymers with differing proportions of L-lysine and L-glutamic acid (Blout and Idelson, 1958; Blout et al., 1961). If the p H of a solution of a copolymer is such that the side chain of one of these species of residue bears no charge and is hence conducive to helix format,ion, the other species of side chain will be charged and thus, if present along the polypeptide chain in sufficient numbers, will tend to disrupt the helix by mutual electrostatic repulsion. One would accordingly expect a set of copolymers with increasing fractions of lysine to exhibit
47 1
OPTICAL ROTATION AND PROTEIN CONFORMATION
progressively lower helical contents at pH 3, at which the poly-L-glutamic acid helix is stable. Infrared absorption spectra indicated the presence of both helical and random regions in solution at a variety of pH's but were not amenable to quantitative assessment. Studies of hydrogen-deuterium exchange have, however, provided information on the degree of intramolecular hydrogen bonding as a function of lysine content at low pH (Blout et al., 1961). The estimates of helical content from this method compare reasonably well with those based on the change in bibs for this same series of copolymers at pH 3, as shown in Table V. The specific rotation changes almost linearly with bibs, thus indicating that the rotatory parameters not TABLEV Comparison of Rotatory Dispersion with Hydrogen-Deuterium Exchange for Partial Helical Content i n Copolymers of L-Glutamic Acid and L-Lysine Polymer Composition (molar ratio, L-glu:L-lys)
lo:o 7:3 6:4 5: 5 4:6 0: 10 ~
Optical rotation*
H-D exchanget
bo
% Helix
% HEAH
-12
-8
-625 -586
>90
100
-27 -51 -69 -92
-439 -310 -131 0
70 50 <25 0
100 65-70 65-70 45 25 -
~~~~
* (Blout and Idelson, 1958.) In aqueous solution, pH 3.
t
(Blout et al., 1961.) D20solution, pD near 3. HEAH: hard-to-exchange amide hydrogen.
only agree with an independent measure of helical content but are also self -consistent. The helical reference conformation of these copolymers cannot be obtained in aqueous solution because of the inherent upper limit imposed by their composition, but the heiical attributes of their constituent residues are both well established and known to be nearly identical. If one nonetheless raised a question as to what scales to adopt for the measures of helical content in these cases, a partial answer may be given by the rotatory behavior of an equimolar copolymer of L-lysine and L-glutamic acid in 2chloroethanol, in which it is completely helical (Doty et al., 1958). Under these conditions, b f is -636, a value almost identical with this parameter for the helical forms of poly-L-lysine and poly-L-glutamic acid in aqueous solution. This agreement is a further example of the relative invariance of bo in the face of solvent changes. The full change in specific rotation at 589 mp, 106", and the value of a?-D,+870, are both somewhat higher than the
472
PETER URNES AND PAUL DOTY
corresponding values in aqueous solution, but precise agreement cannot he expected for these two scales because of the difference in solvent. I n summary, we have therefore seen that poly-L-lysine presents a valuable model for a partmiallyhelical polypeptide chain, one which is amenable to conformational analysis by optical rotatory dispersion. The method by which residues in a helical conformation may be discerned and counted against a background of disordered regions has been illustrated with this polypeptide under almost ideal conditions. The adequacy of the method is corroborated by copolymers a step closer to proteins in complexity, butJ some of the limitations that will be encountered in its application to proteins are already foreshadowed. Before this application is discussed, however, two ot,her phenomena relevant to protein structure that are clearly rxhibited in synthetic polypeptides, the helix-coil t,ransit,ion and t,he /3-conformatlion, will be considered.
H . Helix-Coil Transitions The primary concern of this review is the capacity of optical rotation to detect and measure different conformations of a polypeptide chain and their mixtures rather than the transformations among them. However, inasmuch as the manner in which a helix becomes a random coil is one of its cardinal attributes, the transition behavior of a polypeptide, especially if it is of uncertain conformation, is indeed pertinent to a rotatory interpretation of its static or equilibrium structure. For example, as we have just seen, the way in which the rotatory parameters of poly-L-lysine change with pH is evidence that they truly represent stages in the transition and hence are a measure of helical content. One would expect, moreover, optical rotation to be an extremely sensitive method for following the course of this transition, for it reflects the conformational environment of individual residues rather than over-all changes in molecular shape. Changes in molecular shape as detected by hydrodynamic methods demonstrate that a helix-coil transition has in fact occurred, but in a comparison of hydrodynamic and rotatory data, as has been carried out for poly-y-benzyl-L-glutamate (Doty and Yang, 1956; Doty, 1957), poly-L-lysine (Applequist and Doty, 1961), and poly-P-benzyl-L-aspartate (Bradbury et aE., 1960b), optical rotation displays well-defined changes as opposed to the more gradual, unsymmetrical alterations in hydrodynamic properties. For this reason, it is one of the most widely used means for determining the solvent, pH, and thermal dependence of transitions involving helical macromolecules, including collagen and polynucleotides (Doty, 1956, 1957; Fresco, 1961). Helix-coil transitions have been followed by optical rotation for almost all high molecular weight polypeptides hitherto discussed, usually in the course of delineating the conditions under which the helical and disordered
OPTICAL ROTATION AND PBOTEIN CONFORMATION
473
forms occur. Nearly all transitions, whether accomplished by changes in solvent polarity, pH, or temperature, are centered about a distinct critical point axid exhibit a relatively sharp sigmoidal shape. As will be seen, this sharpness has theoretical significance and in addition can be used as a criterion of conformational change in a variety of experimental contexts, for example, the study of relative stabilizing effects of various side chains. It is perhaps useful in this brief discussion to list the polypeptides in which helixcoil traiisitions have been induced by these several means, as is done in Table VI. A number of theoretical investigations dealing with thermodynamic and statistical mechanical aspects of the helix-coil transition have recently appeared (Hill, 1953a, b; Schellman, 1955, 19588; Zimm and Bragg, 1958, 1959; Gibbs and DiMarzio, 1958, 1959; Rice et al., 1958; Peller, 1959; Lifson and Roig, 196l), and are discussed in a current review by Katchalski and Steinberg (1961) . These treatments, though differing in important details, uniformly produce a transition of characteristic shape in which the sharpness increases with the number of hydrogen bonds that can enter into a cooperative process and thus lead to the correct prediction that high molecular weight polypeptides will display the sharpest transitions (Doty and Yang, 1956). In the context of the theory of Zimm and Bragg (1958,1959), the molecular weight dependence of the thermal transition of poly-y-benzylL-glutamate in ethylene dichloride-dichloroacetic acid mixtures has been studied (Zimm et al., 1959). It is important to note that the transition in this solvent is inverted, for the helical form becomes stable with increasing temperature, a phenomenon which can be accounted for if the entropy of mixing for dichloroacetic acid as it leaves the solvated coil is greater than the entropy lost in the formation of the helix. The sharpness of the transition increases strikingly with molecular weight (Fig. l l ) , and, more significantly, the shape and temperature dependence of each is well described by one function in which the chain length is appropriately varied. The fundamental parameters of this function, one related to the equilibrium constant for adding a residue to a section of helix, s,and the other a measure of the difficulty of starting a helical segment, u, thus in this case appear sufficient to characterize the transition. The theoretical treatment of Zimm and Bragg has also been applied to the shallow thermal transition of poly-L-lysine, in which the variation of helical content as a function of temperature has been obtained from rotatory dispersion measurements (Applequist and Doty, 1961). If one assumes that the parameter u is the same as for poly-y-benzyl-L-glutamate,it is then possible to obtain the heat per residue for the formation of the helix of poly-~lysine. The small magnitude of this result, between -50 and -80 cal/mole near pH 10, as compared to the enthalpy of the hydrogen bond estimated
TABLE VI List of Helix-Coil Transitions i n Synthetic Polypeptides Studied by Optical Rotation* Polypeptide Poly -L-alanine Poly-fl-benzyl-L-aspartate
Poly-r-benzyl-L-glutamate
Poly -1-benzyl-L-histidine Poly-d-carbobenzoxyL-lysine Poly-0-carbobenzoxy-Ltyrosine Poly-r,-glutarnic acid
Poly-L-histidine Pol y-L-leucine Poly -L-lysine Copoly-L-lysine-L-glutamic acid
Poly-L-methionine Copoly-L-methionine-Lvaline Copoly -L-methionine-Smethyl -L-cysteine Poly-L-tryptophan Poly -L-tyrosine Copoly-L-tyrosine-Lglutamic acid
Transition variable
Reference
CHCla: DCA:TFA CHCla: DCA CHCIJ: DCA; m-cresol: DCA CHCla: DCA CHC1s:DCA
Fasrnan (1961a) Karlson et al. (1960) Bradbury et al. (1960b)
CHC13: DCA CHC1a:DCA CHCla: DCA Temperature Temperature Temperature CHCI,: DCA CHCla: DCA CHCl,: DCA 2-Chloroethanol: TFA
Blout et al. (1957) Karlson et al. (1960) Fasman (1961a) Doty and Yang (1956) Zimrn et al. (1959) Calvin et al. (1959) Norland et al. (1961) Fasrnan et al. (1961) Fasman (196la) Katchalski (1958)
PH PH PH PH PH Temperature PH CHC13:TFA PH Temperature 2-Chloroethanol: TFA PH PH CHC1,:DCA:TFA CHCl,: DCA:TFA CHCI,: DCA:TFA
Doty et d.(1957) Idelson and Blout (1958) Imahori and Tanaka (1959) Goldstein and Katchalski (1960) Wada (1960) Doty et al. (1957) Norland et al. (1961) Fasman (1961a) Applequist and Doty (1961) Applequist and Doty (1961) Doty et al. (1958) Doty et at. (1958) Blout and Idelson (1958) Fasman (1961a) Bloom ct al. (1961) Bloom et al. (1961)
CHCl3: DCA:TFA
Bloom et al. (1961)
DMF: DCA DMF: DCA PH PH
Sela et al. (1961) Katchalski (1958) Coornbes et al. (1960) Urnes et al. (1961b)
Fasrnan (1961a) Doty (1957)
* Solvent abbreviations: CHCl, , chloroform; DCA, dichloroacetic acid; DMF, dimethyl formamide; TFA, trifluoroacetic acid. 474
475
OPTICAL ROTATION AND PROTEIN CONFORMATION
by Schellman (1955), - 1500 cal/mole, thus suggests that interactions other than hydrogen-bond formation influence the stability of the poly-Llysine helix. The sharpness of a transition that occurs as temperature, pH, or solvent composition is varied permits each of these three scales to be used as a
I
I
- 10
I 0
I 10
I
20
I
30
I
40
T-TC OC FIG. 11. Comparison of theoretical curves, which describe the thermal helix-coil transition of a polypeptide system as a function of chain length, with experimental points obtained for polyr-benzyl-L-glutamate polymers of varying degrees of polymerization, n. I n this solvent mixture, ethylene dichloride and dichloroacetic acid, the helical form, characterized by the more positive rotations, is stable at higher temperatures. The manner in which increased length sharpens the transition, both in theory and in actuality, is here clearly illustrated. For comparative purposes, T, is defined as the temperature a t the mid-point of the transition for the sample of highest molecular weight. (Zimm et al., 1959.)
measure of relative stability for helical polypeptides that differ in composition, molecular weight, or even the proportion of optical isomers which they contain. In this use of optical rotation to obtain the profile of the transition as conditions are systematically varied, the method resembles a titration. It has been shown, for instance, that as D-residues are introduced into poly-y-benzyl-L-glutamate, the critical concentration of dichloroacetic acid required to disrupt the helix decreases, thus indicating that the helix becomes inherently less stable as the meso-composition is approached. The transition was also found to broaden with increasing amounts of D-residue;
476
P E T E R U R N E S A X D PAIJL DOTY
this finding can perhaps now be interpreted as the consequence of the relative shortness of helical segments with opposite sense (see Sectlion 111, D ). Calvin et al. ( 1!)59) have employed the same thcrmal transit'ion studied hy Yang and Dof,y ( 1956) and Zimm et al. { 1!)5Y) to show that the suhstitution of deuterium for hydrogen in the intramolecular bonds lowers the temperature of transition to the helical form and conclude that deuterium stabilizes the helix. E'asman (l96la) has recently determined an order of relative stability for polypeptide helices in solvent mixtures of chloroform with dichloroacetic acid and trifluoroacetic acid in which he has followed the transitions by the change in bo ,a procedure which minimizes the effect,s of solvent alteration upon conformational measurement. I n terms of critical concentration of organic acid, the least stable polypeptide in his series is poly-P-benzyl-L-aspartate, with poly-t-N-carbobenzoxy-L-lysine, poly-ybenzyl-L-glutamate, poly-L-methionine, poly-L-alanine, and poly-L-leucine following in order of increasing stability. By the same method, Bloom 4 al. (1961) have shown that L-valine, a residue which in its polymeric form assumes only the @-conformationin the solid state (Rlout, c>tal., IYOO), reduces the helical content of poly-1,-methionine by about the same proportion in which it is incorporated, but that the remainiiig helical regions are little affected in their stability. On the other hand, another @-forming amino acid, S-methyl-L-cysteine, does not exert a disruptive effect, on a poly-L-methionine helix until it comprises about 50 % of the residues. The relative influence of the ionizable side chains of glutamic acid and lysine upon helical stability has been estimated from the optical titration of equimolar copoly-L-lysine-L-glutamic acid in aqueous solution ( Doty et al., 1958). Although one expects the disruptive force of positively charged ammonium groups a t low p H to equal the effect of negatively charged carboxylate groups at high pH, there is appreciable helix formation, about 50 %, only a t low pH, that is, where the carboxylate groups are neutralized. Sirice the neut.ralization of the ammonium groups at high pH fails to produce helical regions in the face of negat,ive elect,rost,aticrepulsions, it is peasonable to think that the gluttlmyl side chains can participate in some kind of mutual interact,ion that st,abilizes their helix. The poly-L-glutamic acid helix in fact appears to be more stable than that of poly-L-lysine, for 8 M urea is able to disrupt only the latter (Wada and Doty, 1961; Klemperer and Doty, 1961). That some stabilizing effect, persists into the neutral range is suggested by the rotatory dispersion parameters for this and similar copolymers of lysine and glutamic acid (Blout and Idelson, 1958), which indicate a low but significant helical content. Since neither poly-L-glutamic acid nor poly-L-lysine a t this pH have helical content, the lysine residues may here contribute to helical stability through electrostatic interaction with carboxylate groups. The state of ionization of the side chains can he
OPTICAL ROTATION AND PROTEIN CONFORMATION
477
followed over the helix-coil transition by their titration, as has been done for poly-L-glutamic acid (Doty et al., 1957; Imahori and Tanaka, 1959; Goldstein and Katchalski, 1960; Wada, 1960; Wada and Doty, 196l), poly-L-lysine (Applequist and Doty, 1961 ), and their equimolar copolymer (Doty et al., 1958), so that the manner in which they influence helical stability and are in turn affected in their ionization by conformational changes can be investigated. Since the stability of intramolecular hydrogen bonds in water is marginal, as pointed out by Harrington and Schellman (1956), the various amino acid side chains that occur in proteins may be decisive factors in determining whether a segment of given composition is helical. The basic problem, however, of the manner in which residues of diverse chemical nature promote helical stability in water is only beginning to become clear. Recent work upon a helical poly-L-alanine block made soluble by copolymerization wit,h glutamic acid indicates that this helix cannot be disrupted in wat,er (Doty and Gratzer, 1961). If 30 % L-alanine is randomly introduced into L-lysineL-glutamic acid copolymers similar to those described above, then helical content at neutral pH is increased to over 40 % as judged from a bo of -26% (Priedman and Doty, 1961). Since alanine does not have this effect with either of these residues alone, this finding suggests that it enhances helical stability by favoring the interaction of glutamic acid and lysine as well as by its own intrinsic stabilizing influence. Studies of helix-coil transitions in nonaqueous solvents and with homogeneous polypeptides must, of course, be applied to proteins with caution, both because side chains such as that of poly-P-benzyl-L-aspartate do not naturally occur and because a side chain conducive to instability among its own kind, serine, for example, may coexist well with other species in the aperiodic sequences typical of proteins.
I . Rotatory Properties of the fl-Conformation In contrast to the helical conformation, the rotatory behavior of the @form has received until recently only sporadic attention. Perhaps the principal reason for this neglect is that the @-formhas become associated with insoluble structural proteins rather than biochemically functional molecules and is encountered in the latter class most obviously as an aggregated product of denaturation. The conditions which govern its appearance in synthetic polypeptides have furthermore precluded any assurance that one is dealing in solution with a single conformation, for it exhibits a multiple dependency upon concentration, solvent polarity, and chain length that can involve it simultaneously with helices and random coils. The dependence on concentration (Blout and Assadourian, 1956; Yang and Doty, 1957; Wada et al., 1961 ) is an attribute of any intermolecularly bonded aggregate which, if the equilibrium is shifted to favor the @-conformationover
478
PETER URNES AND PAUL DOTY
the random coil, may lead to unmanageable precipitation. Inasmuch as the p-form is stabilized by hydrogen bonds, its formation is enhanced by nonpolar solvents (Yang and Doty, 1957; Fasman and Blout, 1960; Wada et al., 1961), but since this trait is shared by the helix, it is principally the shorter polypeptide chains that have been employed for characterization (Ambrose and Elliott, 1951a; Mitchell et al., 1957). The p-conformation has also lacked the theoretical stimulus afforded the a-helix. In the single effort in this direction, Murakami (1954) calculated the optical rotation at the sodium D line of short extended chains of poly-L-alanine by combining the polarizability treatment of Kirkwood ( 1937) with the one-electron theory of Condon et al. (1937), but there is no reason to suppose that the data with which he compares his results represent the p-conformation. The geometry of the various extended possibilities is known (Pauling and Corey, 1951) and a means for their distinction by infrared analysis is now at hand (Miyazawa and Blout, 1961). Attempts to derive the rotatory dispersion of peptide bonds in that variety of extended form which actually occurs by using the theories of Moffitt et al. (1957) and Fitts and Kirkwood (1956a) might therefore be undertaken. It is just ten years ago that Robinson and Bott (1951) noted the first correlations between optical rotation and polypeptide conformation. They observed that the specific rotation of copoly-~-methyl-L-glutamate-DLphenylalanine in m-cresol became rapidly more positive with increase in chain length and leveled off at a constant value at high molecular weights, a change paralleled by an increase in intrinsic viscosity. By contrast, in formic acid only a slight decrease in specific rotation occurred over the same range of molecular weight. The infrared spectra of films cast from m-cresol revealed, in terms of the characteristic absorption frequencies, the presence of both a- and p-forms with the proportion of a increasing with molecular weight, while all films cast from formic acid contained predominantly the p-form. If their conclusion, that the change in specific rotation in m-cresol is determined by the proportion of a- to &form, is correct, then from this and a similar correspondence between optical rotation and infrared evidence upon formic acid films, one might infer that the &form is characterized by rotations considerablymore negative than those of the helix. However, in the light of more recent work which shows that the &conformation has substantial positive rotations, this first interpretation of optical rotation in terms of chain conformation may be seen to illustrate the pitfalls in dealing with molecular aggregates. It is most probable that the negative rotations here represent random coils which, as the solutions were concentrated to films, entered into p-structures. From the vantage point of the knowledge gained in the intervening years, this early study nonetheless clearly contains the essential correlations of the helical conformation with its rotatory, hydro-
OPTICAL ROTATION A N D PROTEIN CONFORMATION
479
dynamic and molecular weight properties, and brings out as well the positive rotatory contribution of the helix by D-L cancellation. Three separate studies on synthetic polypeptides have shown that the rotatory dispersions of solutions containing a large proportion of the @-conformation, as judged from their infrared spectra, are more positive than those for random coils and become increasingly so as the wavelength is decreased into the near ultraviolet spectrum. Both Yang and Doty (1957) and Wada et al. (1961) formed the@-structureby using low molecular weight poly-y-benzyl-L-glutamate in nonpolar solvents. Since poly-0-acetyl-Lserine does not appear to form helices even under favorable conditions, Fasman and Blout (1960) were able to obtain the @-formof this polypeptide for relatively long chains of about 125 residues. However, the character of the dispersions in these two cases is different, for poly-y-benzyl-L-glutamate exhibits complex dispersion that fits a Moffitt plot while poly-0acetyl-L-serine gives simple dispersion with A, of 214 mp and a consequent bo value of zero. It is perhaps too early in the study of the @-formto reconcile these findings, but several important differences may be pointed out. As the @-conformation of poly-0-acetyl-L-serine is formed from the random coil in dichloroacetic acid-chloroform mixtures, the specific rotation continues to rise with decrease in solvent polarity, but because of gelation no dispersion measurements could be made on the samples presumably richest in @-content. Since the random coil of this polypeptide is unusual in having a positive dispersion characterized by a A, of 120 mp in dichloroacetic acid, a background provided by this peculiar curve might mask features of the positive @-dispersionthat would otherwise emerge if this conformation were present in higher concentration. Negligible bo values have also been reported for polymers in films with some @-content.Elliott et al. (1958) found that poly-DL-alanine composed of two-thirds D-residues, a polymer that gave infrared evidence of mixed @-formand random coil, and Bombyx mori silk cast from dichloroacetic acid both had bo values of about zero. On the other hand, the dispersions obtained by Yang and Doty (1957) for poly-y-benzyl-L-glutamate in a mixture of @-formsand coil forms did not obey the simple Drude equation, and Imahori’s analysis of these data yielded positive Moffitt slopes and intercepts after corrections had been made for the intrinsic residue contribution (Imahori, 1960). Imahori has moreover found that denatured bovine serum albumin and ovalbumin in solution display positive bo and corrected a0 values in sharp contrast to the negative slopes characteristic of the native proteins and has in addition been able to correlate the positive slopes with the @-formin the protein precipitates by infrared spectra. Wada et al. (1961) have recently carried forward this suggestion that the @-conformationdisplays complex disper-
480
PMTEH URNMS AND PAUL DOTY
sion fitting a Moffitt plot and characterized by parameters, a I Dand b: , that are functionally analogous to those for the helix. Employing low molecular weight poly-7-benzyl-L-glutamate with a chain length of about eleven residues in dioxane and ethylene dichloride, they have attempted to measure the fraction of &conformation in solution from its infrared spectrum and then, accepting the coefficients established for the helix and random coil of this polymer, to compute the Moffitt parameters for the /?-form.An expression for the optical rotation of a mixture of helices, /?-aggregates,and disordered regions can be derived in much the same way as Eq. (33) if the same Xo fits the dispersion of each form. One obtains the equation
in which constants and a fraction appropriate to the &form have been introduced. If these constants are known, this equation implies that the relative proportions of all three conformations can be simultaneously determined from the observed slope and intercept of one dispersion. The possibility of gaining this much information from one dispersion emphasizes that for the simpler mixture represented in Eq. (33), either the slope or the intercept alone is sufficient to apportion the helical and disordered conformations. On the assumption that the helical content in their system is constant while @-aggregatesare formed a t the expense of random coils, Wada et al. are able to specify the ratio of b! to a{-Dand thereby estimate their individual values to be +840 and +420, respectively. Their work thus agrees qualitatively with that of Imahori in indicating that the /?-conformation is characterized by a positive bo in contrast, to the negative value for the helical form, whereas a positive a. is an attribute of both structures. Imahori's measurements (Imahori, 1960) on denatured bovine serum albumin indicate that the Moffitt parameters for the p-form in aqueous solution, a t D = +234 and b{ = +250, are smaller than those found by Wada et al. in organic solvents, so that it would obviously be desirable to characterize the rotat,ory properties of this conformation in water more thoroughly by taking into consideration the simultaneous presence of helical and random conformations. The equilibrium among these three conformations in both organic and aqueous media is complex, for it is a common observation that as solutions of high molecular weight polypeptides are slowly adjusted to bring about a helix-coil transition, a transient haze representing 0-aggregates often develops (Imahori, 1960; Klemperer and Doty, 1958). Although formation of /?-aggregateswith proteins appears in only a few cases to have been reversed, as, for example, in the studies of Ambrose and Elliott (1951b) and Imahori (1960), these observations upon synthetic polypeptides suggest that the equilibrium between helical and p-conforma-
OPTICAL ROTATION AND PROTEIN CONFORMATION
48 1
tions, one which must proceed through the intermediate of a random coil, is easily reversible. Since poly-L-lysine is susceptible to @-formationupon heating in solution (Applequist and Doty, 1961; Rosenheck and Doty, 1961) and can be reconverted from the precipitated form to the helical conformation, it may be a suitable polypeptide for further rotatory characterization of the @-conformationin aqueous solution. In addition to appropriate conditions of molecular weight, solvent polarity, and concentration, side chains that interfere with helix formation can also influence the appearance of the @-form.This is perhaps the cause of the failure of poly-0-acetyl-L-serine to adopt the helical conformation and instead display a rather sharp solvent induced transition on going from the random coil to @-aggregates(Fasman and Blout, 1960). That this effect is also operative in the solid state is indicated by the recent investigation of Blout et al. (1960) which shows that some synthetic polypeptides, poly-Lvaline and poly-S-methyl-L-cysteine, for example, display only the @-structure in films. This suggests that either steric or thermodynamic factors may in these cases preclude helix formation. Humidity is also a factor in determining the appearance of the &form in the solid state, for in oriented films of poly-L-lysine (Blout and Lenormant, 1957;Elliott et al., 1957a) and poly-L-glutamic acid (Lenormant el al., 1958), low humidity brings about a reversible transformation from helices to @-structures.Studies on the solid state may be relevant to events at the interior of a globular protein, but as in solution, the irregular sequences in proteins may minimize side-chain effects which promote @-formation. Although the characterization of the p-form by rotatory dispersion requires further work, the information that is available indicates that it can be distinguished by its rotatory properties from pure forms of other conformations, helices of both senses and disordered chains, in solution. The capacity of the method to assess mixtures of this conformation with helices and disordered regions, at3 may occur in certain proteins, will be given further consideration in Section IV, G.
IV. THE OPTICALROTATORY DISPERSIONOF PROTEINS The aim of this final section is twofold. It will first examine the basis upon which the rotatory dispersions of proteins may be treated in the same manner that has been employed for synthetic polypeptides and, secondly, assess the extent to which the dispersive properties of proteins may be quantitatively explained in terms of known conformations of a polypeptide chain. A pattern of conformational analysis embodying the hypothesis that a polypeptide chain is partitioned into standard helical segments and disordered regions has been shown capable of a rather precise estimate of partial helical content in polymers such as poly-L-lysine and copoly-~-
482
PETER URNES AND PAUL DOTY
lysine-L-glutamic acid. However, the value of this approach for more intricately structured protein molecules remains to be shown. The following discussion will focus upon the plausibility of this simple analysis in view of the similar dispersive features displayed by both proteins and synthet,ic polypeptides and then will comparc the results of its application to proteins with independent estimates of partial helical content. It will be seen that the structure of many globular proteins can be understood in terms of this model, but with the increasing number of exceptions that cannot be accommodated by it, the occurrence of the @-form,helices of opposite sense, and other chain arrangements must on occasion be considered.
A . The Compact, Rigid Structure of Globular Proteins Globular proteins exhibit hydrodynamic behavior which poses, rather than illuminates, the problem of their secondary structure. Quite unlike synthetic polypeptides, for which hydrodynamic evidence is a critical link relating optical rotation to conformation, native globular proteins are neither rodlike molecules nor flexible coils. They behave instead as rigid, moderately asymmetrical particles with dimensions sufficiently small to require that their polypeptide chains are folded in some compact manner (Edsall. 1953). That they are rigid in construction is shown by the relative insensitivity both of their viscosity to changes in pH and ionic strength and of their optical rotation to small changes in temperature. Whereas the viscosity of a randomly coiled polyelectrolyte has a marked dependence upon pH and ionic strength, globular proteins such as bovine serum albumin assume this property only when denatured, otherwise maintaining a constant, comparatively low viscosity (Yang and Foster, 1954; Tanford, 1958). Schellman (1958a-f) has found that the absolute magnitude of the rotatory power for native globular proteins like P-lactoglobulin, ovalbumin, and insulin is constant or increases slightly with a rise in temperature short of denaturation, while for proteins already denatured the temperature coefficient of optical rotation is markedly negative. According to the rule of Kauzmann and Eyring (1941), steric restraint enhances the intrinsic rotatory power of a molecule by preventing an averaging out of configurations through free rotation about one or more of its bonds. It follows that the rotatory power of molecules possessing this restraint should have relative thermal constancy, whereas those lacking it and hence susceptible to the increased freedom of orientation resulting from a rise in temperature should exhibit a diminished rotatory power. The small temperature coefficients of optical rotation for native globular proteins, as compared to the large negative values for their denatured chains, thus argues for a relative rigidity of their structure. The compactness with which the polypeptide chains
OPTICAL ROTATION A N D PROTEIN CONFORMATION
483
must be folded into native globular proteins in solution receives support from the size of their crystalline unit cells together with the fact that small ions are capable of penetrating the hydrated lattice but not the actual protein molecules (Bragg and Perutz, 1952). The inpenetrability of globular proteins in solution to small ions can be further argued from the response of titrable groups to changes in ionic strength (Tanford, 1955).
B. Evidence for the Existence of Helices in Globular Proteins The presence of a-helical segments in native globular proteins would provide some structural basis for their folded nature, for this conformation offers a highly compact and rigid manner of folding a polypeptide chain. It is therefore of cardinal importance that the first structure determination of a protein by X-ray crystallography, that of Kendrew on sperm whale myoglobin reaching a resolution of 2 A (Kendrew et al., 1960, 196l), has established that 77% of the polypeptide chain in this globular protein is incorporated in right-handed a-helical segments. The essential similarity of this molecule to hemoglobin at the 6 A resolution achieved by Perutz (Perutz et al., 1960) for this latter protein makes it almost certain that hemoglobin also contains a large proportion of a-helix. The myoglobin model based on electron densities shows that the helical portions are connected by convoluted sections without obvious order and that they are packed to give an over-all ellipsoidal shape which is in rough agreement with the dimensions of horse heart myoglobin in solution as calculated from its hydrodynamic properties (Edsall, 1953). Besides permitting a count of the number of residues in helical array, X-ray diffraction yields information on the distribution and mutual orientation of helical segments that no other method as yet promises to supply. The amino acid sequence of myoglobin is in large part known, both from the crystallographic indentification of side chains (Kendrew et al., 1961) and from chromatographic techniques (Edmundson and Hirs, 1961), so that direct evidence is now available concerning the side chains that can coexist in a helix. For example, X-ray and chemical methods indicate that both serine and aspartic acid, residues which may hinder helix formation in their polypeptides, occur in helical segments. Although it is not certain how much the helical segments contribute to the total stability of myoglobin and hemoglobin, these studies demonstrate that a-helices can exist in globular proteins and in these instances can account for much of the folded character of the molecules. Although we must anticipate the discussion of optical rotatory dispersion as applied to proteins, it is relevant to mention here the ultraviolet measurements upon myoglobin which have already been described in connection with the issue of helical sense (see Section II1,D). The agreement of these measurements upon myoglobin in solution with helical content in the crys-
484
PETER UHNES AND PAUL DOTY
t,al is good evidence that the secondary structure of this protein, consisting of right-handed a-helices, persists in solution. The estimate of 70 to 80% helix in terms of bo and the optical rotation at 233 mp (Beychok and Blout, 1961; Urnes et al., 1961a) receives support from far ultraviolet spectra that indicate a hypochromism at 190 mp compatible with more than 70 % helical content (Rosenheck and Doty, 1961 ) , although hydrogen-deuterium exchange studies suggest a somewhat lower value, about 50% (Benson and Linderstram-Lang, 1959). Beychok and Blout ( 1961) have also measured the ultraviolet dispersion of hemoglobin in solution, with a like estimate of 75-80 % helical content. The concordance of rotatory dispersion with known helical content in the crystals of these two proteins thus offers an answer to a fundamental methodological question: to what extent are structural determinations by X-ray crystallography and companion studies of amino acid sequence relevant to the functioning of proteins in solution? Since these results suggest that the intricate architecture of myoglobin and hemoglobin is largely preserved in solution, one can perhaps now undertake to explain the biological activity of these globular proteins in terms of a complete molecular structure. Infrared evidence for the existence of intramolecularly bonded structures in globular proteins has been obtained by Elliott et al. (1950) and Ambrose and Elliott (1951b) who studied suspensions of crystals and dry powders of insulin, chymotrypsinogen, glyoxalase, and hemoglobin. Bands associated with the a-form shifted to those characteristic of the p-form upon heat denaturation, but the a-bands of insulin and chymotrypsinogen as well as the hormonal activity of insulin could be regenerated by treatment in phenol or m-cresol. I t is now known that the helical conformation can be distinguished from the random coil only with difficulty at the 1660 cm-' absorption band (see Section 111, A , 2), but the fact that these authors were able to reproduce the band after treatment similar to that which promotes helix formation in synthetic polypeptides may argue for the existence of helical regions in these proteins and their necessity for the activity of insulin. Although other infrared studies have been carried out on globular proteins (Uzman and Blout, 1950; Chouteau, 1953; Lenormant and Blout, 1954; de Lozts and Lenormant, 1956), it is in general true that the helical conformation cannot be detected with certainty and that the high solvent absorption of water in the vicinity of the critical bands confines the investigation of proteins to high concentration in either deuterium oxide or nonaqueous media. Two other methods, hydrogen-deuterium exchange and far ultraviolet spectroscopy, have produced further evidence that some globular proteins in solution are partially helical. Since quantitative estimates of helical coiitent may be made from these measurements, a discussion of their re-
OPTICAL ROTATION AND PROTEIN CONFORMATION
485
sults will be deferred for comparison with optical rotatory dispersion. It is sufficient to point out in this context that slowly exchanging hydrogen atoms are compatible with any sequestration of amide groups from the solvent, including hydrogen bonding in structures other than helices. I n contrast, the hypochromism associated with the peptide absorption band a t 190 mp is, so far as is known, a more direct reflection of the helical conformation.
C. Points of Similarity between Protein and Polypeptide Dispersions The optical rotatory dispersions of all native globular proteins thus far measured have obeyed the simple Drude equation in the spectral range iisually encompassed, 350400 mp. I n most cases, X, values vary from 230 to 280 mp, although a number of proteins have X, values below 230 mp and some as low as 180 mp. Optical rotations, which are usually reported as the specific rotations a t 589 mp, range from about -20 to -80". Upon denaturation, all dispersions remain simple, but A, values shift to the more narrow range of 210-230 mp and the specific rotation becomes more levorotatory to a range of about -80 to - 120". Globular proteins for which dispersion data are available have, following the procedure of Schellman and Schellman (196l), been assorted into three groups distinguished by the relation of the native value for X, to the dispersion constant that is usually found for denatured proteins. Thus globular proteins with A, greater than 230 mp appear in Table VII, those with A, in about the same range as denatured proteins, 210-230 mp, in Table VIII, and finally those with A, less than 210 mp, in Table IX. A similar tripartite classification has been made by Jirgensons ( 1961a) with slightly different demarcation points. Tables of data for proteins have been compiled by Schellman and Schellman (1961) and by Jirgensons (1961a) and for both proteins and synthetic polypeptides by Blout (1960). Todd's list of references pertinent to individual proteins and polypeptides is useful (Todd, 1960). The data collated here are representative values for any given protein under physiological and denaturing conditions; hence for a more complete set of values on a protein the references appearing in the same entry should be consulted. The proposal to assimilate the rotatory dispersions of proteins to those of synthetic polypeptides is, in barest form, the recommendation to treat these same data by the Moffitt expression despite the fact that they can be described by the simple Drude equation. Thus one proceeds t o plot [ m ' ] ~ . (A2 - X i ) / X i against Ai/(X2 - X i ) with Xo set to 212 mp, and thereby obtains an intercept, aiba,and a slope, bibs, which can be given conformational interpretations through Eqs. ( 3 5 ) and (36). These parameters, which are in general cited simply as a0 and b, when their experimental origin is
486
P E T E R URNES AND PAUL DOTY
TABLEVII Optical Rotatory Properties of Globular Proteins with Native A, Values Greater Than 2.90 mp* ~
Solvent
Protein
Optical rotation
XO
mr)
ao
bo
Reference
Albumin, bovine serum
PH 5
284
Yang and Doty (1957)
H a
-
8 M Urea,
-
pH 6.47 8 M Urea, pH 6.6
265 221
8 M Urea 0.1 M KCI, pH 6
-
Doty (19.57); Imahori and Doty (19.57) Doty (1967); Imahori and Doty (1967) Schellman (1968d) Scbellmen (1968d) Imahori (1960) Imahori (1980) Leonard and Foster (1961) Stauff and Jaenicke (1981) Jirgensons (1958a)
Ha0
Albumin, bovine serum, oxidized Albumin, human serum
pH 9.0,p 0.0
213
pH 6.0
267
pH 7.8
262
M Guanidine thiocyanate pH 7.8
226
0.02 M PO4 , pH 8
248
0.1 M PO4, pH 7.5
250
4 M Urea
210
0.07 M P o d , pH 7.6 0.07 M P o 4 , pH 7.6, 6 M urea 0.02 M PO4, pH 8.6
262 223
0.02 M P o 4 , pH 1.9
220
0.01 M NaOH, pH 8.7
243
2.0 M NaBr, CaClz,
273
4
Albumin, human serum, oxidized Alcohol dehydrnKenaae
Aldolase
a-Amylase, pancreatic Amylase, bacterial
@-Amylase Taka-am ylase Taka-amylase A
Arachin Carboxypeptidase
-
221
278
Jirgensons and Ikenab (1959) Jirgensons and Ikenaka (1959) Jirgensons and Ikenaka (1969) Jirgemons (19.59~) Ulmer and (1981) Ulmer and (1961) Sund (1960) Sund (1960)
Vallee Vallee
Jirgensons (1969c, 1961b) Jirgeensons (1969c, 196lb) Jirgensons (1959~)
219
Jirgensona (1968c, 198lb) Jirgensons (1981d)
277 271
Jirgensons (1969~) Jirgensona (1969~)
p H 7.16
293
10 M Urea
221
Glycine buffer, p 0.1, p H 8.7 Dil. NaOH, pH 9.4
232
Maeda and Oikawa (1969) Maeda and Oikawa (1969) Jirgensons (1968a)
269
Jirgensons (1969~)
pH 7.8 Guanidine thiocyanate 0.1 M PO4 , p H 8.1 0.02 M Calcium acetate, pH 7.0
_ _ ~ -
-
TABLB VII-Continued -
Solvent
Protein
Reference
kp:
bo
241 220
-
Schellman (1958f) Schellman (1958f)
235 218
-
Jirgensons (1959~) Jirgensons (1981d)
a-Chymotrypsin
0.1 M NaCI, p H 3 0.1 M NaCI, p H 3,s M urea 0.1 KCI, HCI, p H 3 2 M Guanidine thiocyanate 6-Chymotrypsin pH 3.12 7-Chymotrypsin p H 3.3 a-Chymotrypsin 0.05 M POI, 0.1 M @phenylpropionate, p H 7.7 8 M Urea. p H 7.7 Chymotrypsinogen 0.1 M KCI, p H 8.73 0.1 M KCI, pH 8.5,8 M urea 0.1 M KCI, p H 3.1 2.8 M Guanidine thiocyanate 0.05 M POI, 0.1 M 6phenylpropionate, p H 7.7 8 M Urea, pH 7.7 Clupein chloride 0.1 M KCl, p H 6 Deoxyribonuclense 0.1 M NaBr, p H 3.5 0.2 M POI, p H 7.4 Fetuin 0.2MP01 ,pH7.4,8M urea Fibrinogen 0.6 M KCl
@-Globulin, metal binding Glncagon
Jirgensons (1958b) Jirgensons (1958b) Irnahori et al. (1900)
-
Iniahori et al. (1980) Schellman (1958f) SchelLnlan (19581)
239 224 235 209
-
-
-72
-
0.1 M KCI, p H 8.3 p H 6.7 Ficin Folliclestiniuhting 0.25 M NaBr, pH 8.3 hormone Globin (hernoglo- Ha0 bin)
(rnyoglo-
234 237
-
9.5 M Urea
Globin hinl
-
Jirgensons (1959~) Jirgensons (1961d ) Iniahori et al. (1960)
0
201 233 280 225
-
Iinaliori et al. (1980) Schellman (1958e) Jirgensons (190lb) Kay and Marsh (1959a) Kay and Marsh (195Sa)
-
-2lOt
-
-0t
Cohen and Szent-Gyorgyi (1957) Cohen and Szent-Gyorgyi (1957) Yang and Doty (1957) Jirgensons (1959~) Jirgensons (1960h)
256 261 233
-
-
-
-95
8 M Urea
-
-
H10
-
-234
8 M Urea
-
-
pH 7.3
242
-
Doty (1957); Irnahori and Doty (1957) Doty (1957); Iniahori and Doty (1957) Doty (1957); Imahori and Doty (1957) Doty (1957); Imahori and Doty (1957) Jirgensons (1958s)
231
-
Ray and Marsh (1958a)
222
-
Kay and Marsh (1959a)
NL
-170
Jirgensons (198lf)
318
-367
Jirgensons (196lf)
240
-70
Jirgensons (1961f)
0.1 M Glycine, 0.1 M NaC1,O.l N NaOH, p H 10.4 0.1 M Glycine, 0.1 M NaCl, 0.1 N NaOH, p H 10.4, 8 M urea GLutamic acid de- 0.02-0.10 M so4 or hydrogenase POI, p H 6.9-8.6 0.024.10 M SO4 or PO*, p H 9.5,30 min 0.02-0.10 M S O 4 or Po4, p H 9.5.4 hr
__
487
TABLE VII-Continued -
Solvent
Protein
Optical rotation
Xe
mr)
__ bo Reference
Glycernldeh yde phosphate dehydrogenase a-Glycoprotein Histone
Insulin
Insulin, oxidized A chain
Lactic acid dehydrogenase
p -Lactoglobulin
Lysozytrie
Malic dehydrogen.
am Mcrcaptalhnriiin, bovine Hyoglohin, speriii whale
>H 7.2
238
lirgensona (1959~)
>H5.8
249
H@
-
1 M Urea
-
H20
-
1 M Urea
-
313 3 I M Urea, pH 5.5 ).1 A1 NaC1, p H 7
267 226 228
lirgensons (1958s) Doty (1957); Irnahori and Doty (1957) Doty (1957); Imahori and Doty (1857) Doty (1957); Iniahori and Doty (1957) Doty (1967); Irnahori and Doty (1957) 3chellman (1858~) 3chellnian (19680) 3chellrnan (195Be)
1.1 M NaCI, p H 7.6 M urea 1.8 M (NHdsSOr, p H 5.6 1.2 M P04,p H 3.8
225
3chellriian (1958e)
264
1.1 M NaCl, p H 5.5 1.1 M NaCl, p H 5.5, 8 M urea 1.125 M NaBr, p H 6.2 1.15 M KC1, pH 6.6 5.02 M N(CHd4C1, pH 11.8 1 M NaCl Ha0
249 225
lirgensona (195W 1961b) lirgensons (1059~. 1961b) 3chellnian (1958b) 3chellman (1958b)
222
240 -
264
-
3.25 M NaBr, p H 5.6 1 M Guanidine thiocyanate HzO
254 215
8 M Urea
143
PH 5
266
Joyce and Grisolia (1961) Joyce nnd Grisolia (1961) Jirgensons (1958n)
0.1 af PO^, pi1 7.0
-
Urnes e l al. (19614
220 270
Urnes e l a!. (1061a) Yang and Doty (1957) Doty (1957); I n d i o r i and Doty (1957) Doty (1857); Iiirahori and Doty (1957) Schellman (1958d) Schellrnan (1058d) Jirgensons (1958~) Imahori (1860) Jirgensona (1959~)
280
pII 4.7 €120
-
8 M Uren,
-
p H 7.1 8.6 M Urea, p H 7.1 0.05 M Glycine, p H 5.3
266 226 266
-
HlO Papain
Yang and Doty (1957) Doty (1957); Iniahori and Doty (1957) Doty (1967); Imahori and Doty (1957) lirgensons (1961~) lirgensons (196ld)
9 tvf Urea
8 $1 Ureu.
Ovalbuinin
lirgensons (1958a) ranford et al. (1960) ranford et al. (1960)
231
0.1 N Acetic acid, p H 3.6
-
488
489
OPTICAL ROTATION AND P R O T E I N CONFORMATION
TABLEVII-Continued
-
Protein
Solvent
I&:
Reference
-
Pepsinogen
Prolactin
Ribonuclease
Ribonuclease, oxi dized Ribonuclease, re duced Salmine sulfate Soinatotropin
Thyrodobulin
Trypsin Trypsin inhibitor, Lima bean I Trypsin inhibitor, lima bean I1 Trypsinogen
0.05 M NaCl, PO. buffer, p H 7.7 8 M Urea Dil. NaOH, p H 8.8 4 M Guanidine thio cyanate Hi0
239
Perlmann (1961)
215 282 217
Perlniann (1981) Jirgensons (1980b) Jirgensons (196Ob)
-
8 M Urea
-
0.1 M KCL, pH 5.4 8 M Urea, pH 5.8
233
Doty (1957); Iniahori and Doty (1957) Doty (1957); Imahori and Doty (1957) Schellman (19580 Schellman( 1968f) Jirgensons (1981b) JirgeMons (198lb)
220 233
0.25 M NaBr. p H 4.9 0.1 M NaBr, NaOH, pH 11.6 0.1 M KCl p H 4.0
238 228
0.1 M KCI
226
White (1961) Harrington and Schellman (1958) White (1961)
pH 8.4 0.25 M NaBr, NaOH, p H 9.5 4 M Guanidine thiocyanate, p H 8.5 0.1 M KNOa, pH 8
222 277
Yang and Doty (1957) Jirgensons (1960b)
221
Jirgensons (196Ob)
9.0 M Urea, pH 9.8
225$35 212
0.1 M KCI, p H 5.2 0.1 M KCI, pH 1.3 pH 7.2
235 217 258
Edelhocb and Metsger (1961) Edelhoch and Metzger (1961) Jirgensons (1959~) Jirgensons (1959~) lirgensons el al. (1960)
pH 7.5
280
Jirgensons et al. (1980)
232
Jirgensons (1959~)
3.1 N KCI, pH 3.2
224
-
* Measurements of an and bo are based onXo = 212 mp. All solvents are aqueous solutions. t Based on XO = 210 mp. $ Based on Xo = 216 m p .
clear, are accordingly reported in Tables VII-IX for cases in which this procedure has been fallowed. They are listed as well for a group of fibrous proteins which display simple Drude behavior only when denatured, Table X. It is true that this procedure appears to risk the introduction of pseudocomplexity into data that are quite adequately described by the simple Drude equation, as has been found for random coils in which A, does not equal A0 (see Section 111, C, 1 ) . The application of the Moffitt form therefore requires justification, one which will be attempted by first enumerating points of similarity between protein and polypeptide dispersions and then showing how empirical correlations developed for the Drude equation
490
PETER URNES AND PAUL DOTY
solely within the context of protein structure can receive interpretation in terms appropriate to synthetic polypeptides. 1. Denatured Proteins and Random Coils
I t is not surprising that denatured proteins display the same simple dispersive properties as randomly coiled synthetic polypeptides, for denatured TABLEVIII Optical Rotatory Properties of Globular Proteins with Native A, Values between 2f 0 and 250 mfi*
-
Solvent
Protein
XC
Reference
mrl
a-Casein &Casein EItutnse Luteinizing hormone
Glycine buffer, 8 0.1, pH 8.9 Glycine huffer,p 0.1, pH 9.2 0.05 M Acetic acid, p H 3.4 0.1 M Acetate, pH 5.0
223
Jirgensons (1958a)
218
Jirgensons (1958b)
228
Jirgensons (1961b)
218
Jirgensons (1980b)
pH 9.7
216 229
Jirgensons (196Ob) Jirgensons et al. (1960)
pH 5.24
216
pH 10.9
222
0.1 N Acetatebuffer, pH 4.8 0.1 N Acetate buffer, pH 5.3, 8 M urea, 85°C H20 0.125 M NaBr, p H 4.7 GIycinebuffer.8 0.1, pH 10.76 0.25 M NaBr, pH 6.0 0.25 M NaBr, NaOlI, pH 10.4 pH 7.6
216
Jirgensons (1958b, 19590) Jirgensons (1958b, 1959~) Perlmann (1959)
230
Perlmann (1969)
225 207
Doty (1958) Jirgensons (19588,b) Jirgensons (1958s,b)
210 222
Jirgensons (19680) Jirgensons (19580)
217
Jirgensons et al. (1960)
7
Ovoniucoid, trypsin inhibitor Pepsin
Pliosvitin
Rennin
Trypsin inbibitor, soybean
U Urea
* The inea8urninent of ba is based on Xa = 212 mp. All solventa nre aqueous solutions.
proteins give every evidence of having a solvated backbone in both their deuterium exchange properties and in their hydrodynamic behavior. Although denaturing conditions may, in individual cases, fail to unfold the protein completely, as appears to be true of paramyosin (Cohen and SzentGyorgyi 1957; Simmons et al., 1961), the rotatory parameters for these two types of disordered chain, natural and synthetic, usually lie within the same narrow range of variation, and differences in optical rotation between a denatured protein and a typical randomly coiled polypeptide chain are
49 1
OPTICAL ROTATION A N D PROTEIN CONFORMATION
about what one would expect from dissimilarities in amino acid composition. On the assumption that residue contributions in random coils are additive, Schellman and Schellman ( 1958) have accurately computed the specific rotations of silk and gelatin, using only their known composition and mean residue rotations measured in synthetic random coils for each species of amino acid. One can therefore conclude that the rotatory attributes of TABLEIX Optical Rotatory Properties of Globular Proteins with Native A. Values Less than 810 mu*
-
Protein
Optical rotation
Solvent
XC
(mr)
Reference
BencrJones proteins Example 1 Example 2 Example 3 y-Globulins Example
y-Lactoglobulins Macroglobulins, serum Example
Myeloma globulins Exaniple
[a\D-37 to
(Native)
-54 H a0 pH 1.6 HaO pH 2.5 HaO pH 1.55 (Nntive)
180210 178 219
Jirgensons (1961%)
-41.9 -93.4 190 lalsta -51.8 [alrra -75.9 218 [alM8 -54.3 207 220 Clsra -146.1 [aID -44 to 200-55 217 0.175, [=ID -62 207
Jirgensons (195%) Jirgensons (1959b) dirgensons (1959h) Jirgensons (195Sb) Jirgensons (1959b) Jirgensons (1959b) Jirgensons (1961%)
225
Jirgensons (1958~)
205215 19b 206 196 219
Sehclltrian and Sclrellman (1961) Jirgensons (196ln)
206218 212 217
Jirgensons (19618)
La1544 [a1646
NaBr, glycine, p pH 8.4 2 M Guanidine thioeya- [a10 -96 nate, 50°C [ a ] D -68 to (Native) -75 [-ID -30 to (Native) -38 [ a l v s -37 Ha0 2 M Guanidine thioeya- [a1646 -95 nate ["ID -40 to (Native) -45 Ha0 [ U l M 6 -57 2 M Guanidine thiocya- IUIM -106 mite
* All solvents are aqueous solutions.
Jirgensons (1958~)
Jirgenaons (1960s) Jirgensons (I960n)
Jirgensons (I96Oa) Jirgensons (1960%)
-
this reference conformation, the disordered chain, are essentially identical in these two classes of polymer. 2. Fibrous Proteins and Helical Polypeptides
The helical reference conformation in synthetic polypeptides fortunately has a counterpart in a group of fibrous muscle proteins, for one of which, Pinna nobilis tropomyosin, hydrodynamic and light-scatt,ering evidence indicate a helical form. This protein is classified as a globulin by virtue of its insolubility in pure water and yet Kay (1958) has shown that, its structure is most consistently interpreted as a rigid rod 1400A in length with
492
PETER URNES .4NI) PAUL UOTY
an average diameter of 18.5A. However, given a molecular weight of 134,000, this length is somewhat less than that for an equivalent a-helix. While this finding can be rationalized in several ways, as, for example, by TABLEX Optical Rotatory Properties of Fibrous Proteins* Meroniyosin, heavy
Meronl yosin, light fraction I
Myosin
__ 0.6 M KCl
-300t
0.05 hi Urea
-0t
0.6 M KCl
-440t
9.5 M Urea
-0t
0.6 M KCl
-660t
9.5 M Urea
-0t
0.6 M KCl
-370t
9.5 M Urea
--of
Purltinyosin
Tropom yoain, Pinna nobilis Tropomyosin, rabbit
Reference
bo
Solvent
Protein
-380
n
8 M Urea 0.6 M KC1
-6ont
9.5 M Urea
-190
0.6 M KCl, 0.1 M PO,, PH 7 8 M Urea POa-KCl buffer, fi 0.6 PH 7 8 M Urea, pH 7-7.5 0.6 M KC1
-6nn
9.5 M Urea
-220 -650t -
-620t
-0t -552
-
8 M liren 0.6 M KCI, 0.1
PH 7 8 M Urea
M
roc,
-600
0
-_ Cohen and Ysent-Gyorgyi (1967) Cohen and Szent-Gyorgyi (1957) Cohen and Szent-GyBrgyi (1967) Cohen and Ssent-Gyorgyi (1957) Cohen and Szent-Gyorgyi (1957) Cohen and Szent-Gyorgyi (1957) Cohen and Saent-Gyijrgyi (1957) Cohen and Szent-Gyhrgyi (leal) Simmons el al. (1961) Sinimnna el al. (1961) Cohen and Szent-Gyorgyi (1867) Cohen and Szent-GyGrgyi (1957) Simmons e l al. (1961) Giinmnns el at. (1981) Kay and Bailey (1959) Kay and Bailey (1959) Cohen and Ssent-Gyorgyi (1967) Cohen and Szent-Gyorgyi (1957) Doty (1967); Imahori and Doty (1957) Doty (1957); Imahori and Doty (1957) Simmons et al. (1961) Simmons el al. (1961)
* Measurements of ao and bo are bwed on XO = 212 mlr. All solvents are aqueous solutions. NL: nonlinear plot.
t Based on Xo
= 210 mp.
a helix of a smaller axial displacement per residue, about 1.2 A, it is likely that the effective molecular length is shortened by the existence of some flexibility of the sort that occurs in poly-6-N-carbobenzoxy-L-lysineand poly-L-lysine (see Section 111,A, 3, and 111,G, 3). Oriented crystals of Pinna
OPTICAL HOTATION A N D PROTEIN CONFORMATION
493
nobilis tropomyosin exhibit a typical a-keratin X-ray diffraction pattern (Bailey, 1957), and in view of the water embodied in protein crystals, which Bailey (1948) has shown to be as high as 80-90% in the case of rabbit tropomyosin, the environmental change on entering solution is probably not sufficient to warrant a stable helix of other coordinates. As demonstrated by Kay and Bailey (1959)) the complex dispersion of Pinna nobilis tropomyosin has a bo of -650, a value entirely in harmony with a standard helical polypeptide. It, would be surprising if helices of different coordinates had the same rotatory attributes, and the divergent dispersions of poly-Lprolines I and 11, both of which are variants of a polypeptide helix, together with the dispersion of collagen (see Section IV, I), seem to bear this out. I n any event, the bo values for the tropomyosins, paramyosin, and light meromyosin I (Table X) are compatible with molecular helices. The XO value for their Moffitt plots was determined by Cohen and Szent-Gyorgyi (1957) and found to be 210 f 10 mp, thereby providing a parallel both in procedure and result with the work of Moffitt and Yang (1956) for synthetic polypeptides. If these data are plotted with Xo set to 212 mp, bo values will decrease slightly, for example, from -650 to -630, but not enough to permit, more than a small number of disordered residues. If we accept that these fibrous proteins have essentially the same conformation as helical polypeptides, then their investigation yields good evidence that protein helices do exhibit standard dispersion. The dispersion is complex, bo and Xo values are concordant,, and these proteins upon denaturation give A,: values of about 215 mp with a A[m']meof 85 to 90". The value of for Pinna nobilis t,ropomyosin, computed from the measured rotatory paramet,ers (Tanford et al., 1960), is +t,tiO. The negative sign of bo and t,he increase in levorotat,ion upon denaturation shows that these protein helices are of standard sense, now known to be right-handed. Although denatured Pinna nobilis tropomyosin fails to crystallize upon removal of 8 M urea, it,s optical rotatory properties are completely reversible. Like helical polypeptides, rahhit tropomyosin and paraniyosin display Cotton effect features near 2 3 3 mM with about, the same amplitude, again an indication of high helical content (Simmons el al., 1901; see, Section 11, D). Pinna nobilis tropomyosin contains no proline (Bailey, 1957)) which is consistent with a helical molecule. As might be expected from the study of a 5 % copolymer of L-tyrosine with L-glutamic acid (see Section 111, F ) , the presence of small amounts of tyrosine and histidine in this protein has no appreciable rotatory effect. This protein contains relatively large amounts of amino acids that, do form standard helical polypeptides-glutamic acid, 21 %; lysine, 8 %; alanine, 12 %; and leucine, 12.5 %-but it also has 4 3'% valine, 6 % swine, and 13 % aspartic acid. If these residues behaved as they and their analoguesappear to do in synthetic polypeptides, either pre-
494
PETER URNES AND PAUL DQTY
venting helix formation or giving a helix of opposite sense, then their disruptive effect should be evident in the rotatory and hydrodynamic properties of tropomyosin. Difficulties anticipated from the study of synthetic polypeptides both from side-chain chromophores and helical instability thus appear in this group of proteins to be minimal, and the rotatory attributes of the helical reference conformation may be taken as effectively the same for synthetic polypeptides and typical protein chains. 3. Helix-Coil Transitions in Nonaqueous Solvents Another point of resemblance between synthetic polypeptides and proteins, one which has more bearing on their conformational capabilities than upon optical rotation, is that proteins treated like synthetic polypeptides in nonaqueous solvent mixtures traverse a large part of a helix-coil transition. That they become in large measure helical in solvents of low polarity is shown by the appearance of truly anomalous dispersion with negative bo values for silk fibroin in ethylene dichloride-dichloroacetic acid mixtures and the similarity of the B chain of insulin in dimethyl formamide to the dispersion of poly-r-benzyl-L-glutamate (Yang and Doty, 1957). These findings are further supported by dispersion measurements upon a variety of proteins in 2-chloroethanol (Table XIII),many of which show large positive increments in the specific rotation as compared to values in organic acids as well as the appearance of large negative bo values characteristic of high helical content (Doty, 1958). I n some cases, notably those of rabbit tropomyosin, ovalbumin, and silk fibroin, the transitions brought about by continuous variation of polar and nonpolar components in the solvent are sufficiently sharp and sigmoidal in shape to suggest a cooperative effect. The transition for ovalbumin is comparable to that of equimolar copolyL-lysine-L-glutamic acid in the same solvent mixture, 2-chloroethanoltrifluoroacetic acid, as shown in Fig. 12 (Doty el al., 1958). In other proteins, insulin, ribonuclease, and bovine serum albumin, the transition displayed in similar solvent mixtures is broad and is not sharpened appreciably by the breakage of restraining disulfide bonds (Yang and Doty, 1957). Broad transitions of this type could result either from relatively short helical segments, as with synthetic polypeptide chains of small length, or from helical regions with widely varying stabilities brought about by compositional heterogeneity. The presence of proline, disulfide bridges, and other intramolecular restraints can, of course, preclude full helix-coil transitions as well as limit, helical regions to short segments of chain. If water replaces dichloroacet,ic acid or trifluoroacetic acid as the hydrogen-bonding component in mixt,ures of decreasing polarity, then t,he helical content of disordered water-soluble polypeptide chains will increase continuously, as is, for example, the case with equimolar copoly-L-lysine-L-
OPTICAL ROTATION AND PROTEIN CONFORMATION
495
glutamic acid (Imahori et al., 1958). If globular proteins in their native aqueous environment are treated in this same way, some do not, however, display a steady increase in helical content. As dioxune or dimethyl formamide is first added to aqueous solutions of p-lactoglobulin (Tanford et al., 1960), the specific rotations decrease, and the same phenomenon appears, although to a much lesser extent, with ovalbumin (Frenkel and Horn,
-
-
COPOLY L- LYSl NE L-GLUTA
M IC
ACI D
0-
-20 -
Cu1D -40
-
-
-60 -
-80 -
"OO0
20
CHLOROETHANOL
40
60
00
ic
TRIFLUOROACETIC ACID
FIG.12. Helix-coil transitions of equimolar copoly-I,-lysine-L-glutarnic acid and ovalbumin induced by variation i n solvent composition. The specific rotation is plotted as a function of t h e per cent of trifluoroacetic acid in t,he 2-chloroethanoltrifluoroacetic acid solvent system. (Doty et al., 1958.)
1960). The specific rotation of p-lactoglobulin eventually increases and DO decreases as one might expect with increasing proportions of the nonpolar organic solvent, but the early parts of this solvent-induced transition are compatible with a loss of secondary structure, either of the helical or the @-form,or a solventlike change upon the rotatory power of the peptide bond, alternatives that will be discussed in more detail with respect to the rotatory dispersion of this protein (see Section IV, G, 2). A general explanation for this initial effect is that organic solvents may disrupt the hydrogen-bonded matrix of water and thus free nonpolar side chains from the
496
PETER URNES A N D PA1JL DOTY
compressive forces that tend to sequester them from aqueous solution (Kauzmann, 1959). The stability granted either to helical regions or to some other chain arrangement through the clustering of nonpolar side chains in response to these so-called hydrophobic forces can thus be diminished by moderate concentrations of a nonpolar solvent. This phenomenon, which is specific to water, is hence a consequence of the differing solubilities of the diverse side chains present in proteins. The initial decrease in optical rotation found in aqueous solutions of p-lactoglobulin and ovalbumin is not, however, sufficient to differentiate globular proteins from simpler synthetic polypeptides in their transition behavior, for neither ribonuclease nor human serum albumin appear to exhibit it. The specific rotation of ribonuclease in water-2-chloroethanol mixtures becomes steadily less levorotatory as the proportion of nonpolar solvent increases (Weber and Tanford, 1959). In the case of human serum albumin Bresler (1958) and Bresler et al. (1959) find that only progressive increases in specific rotation occur as the concentration of 2-chloroethanol is increased and that this change is accompanied by a steady rise in viscosity and the corresponding axial ratios characteristic of the formation of rodlikc particles. If these proteins do have some initial helical content in water, us can be argued from their optical rotatory dispersion, then it appears that hydrophobic forces are not required for the stability of these regions. Two other transitional phenomena observed in poly-y-benzyl-L-glutamate have their counterparts in proteins. Under appropriate concentrations of denaturing agent, P-lactoglobulin ( Christensen, 1952), ovalbumin (Simpson and Kauzmann, 1953), ribonuclease (lposs and Schellman, 1959), and lysozyme (Icoss, 1960) undergo an inverted thermal transition to a more highly folded form as judged from optical rotation. As with poly-y-benzylL-glutamate in ethylene dichloride-dichloroacetic mixtures (see Section 111, H ) , a thermodynamic explanation in terms of the relative change in entropy for the solvent and protein can be offered to account for the inversion. The stabilizing effect that the substitution of deuterium for hydrogen produces in the poly-y-benzyl-L-glutamate helix (Calvin el al., 1959) appears to have its analogue in the thermal denaturation of ribonuclease (Rermans and Scheraga, 1959), for this transition to an unfolded form occurs in deuterated ribonuclease at a higher temperature than in the hydrogen-containing protein. This similarity clearly links denaturation in this protein to a process involving the breakage of intramolecular hydrogen bonds.
4. Changes in the Specific Rotation upon Denaturation Although globular proteins can develop helical content in nonpolar solvents, this behavior does not entail that they contain helical segments in their native, aqueous media. Yet the changes in specifir rotation that
OPTICAL ROTATION A N D PROTEIN CONFORMATION
497
attend the loss of their folded structure by denaturation in aqueous solution are strikingly similar to those following the disruption of a helix in a standard synthetic polypeptide. It is well established that the specific rotation in the visible spectrum becomes more levorotatory as the native strdcture is unfolded by changes in pH, urea concentration, or temperature, and that it accompanies other physical measures of the conformational change. As an example of pH denaturation, both Yang and Foster (1954) and Sterman and Foster (1955) have shown that the change in rotation of bovine serum albumin coincides with its reversible expansion below pH 4 as indicated by viscosity, and observations of the rotatory change obtained by Almquist and Greenberg (1934), Jirgensons (1952), and Golub and Pickett (1954) correlate well with the structural changes observed by Pedersen (1953), who followed the sedimentation, diffusion, and viscosity of this protein over the same pH range. To choose only one from among the great variety of proteins that he has studied by optical rotation and viscosity, Jirgensons (1954) has shown that the levorotation and viscosity of 7-globulin increase concurrently a t extremes of pH. Urea denaturation of bovine serum albumin and ovalbumin likewise produces a concomitant increase in levorotation and viscosity, as found by Simpson and Kauzmann (1953), Kauzmann and Simpson (1953), and Frensdorf et al. (1953a, b). LQnis (1956) has shown a similar correlation for lysozyme, as have Steven and Tristram (1959) again for ovalbumin. That optical rotation follows the loss of enzymatic activity as well as the increase in viscosity is clearly illustrated in the urea denaturat,ion of 6-chymotrypsin as studied by Neurath et al. (1956). Schellman (1958c, d, f ) has followed the thermal denaturation of insulin, bovine serum albumin, chymotrypsin, chymotrypsinogen, and ribonuclease as well as their behavior in urea. He finds especially sharp thermal transitions in the optical rotation, exemplified by that for insulin (Fig. 13), that correlate with a n unfolding process as measured both thermodynamically and by the different temperature coefficients of the specific rotation for the native and denatured states. These and numerous other studies demonstrate that optical rotation is a sensitive indicator of structural change and bear out the original interpretation by Kauzmann and Eyring (1941) that rotatory alterations during prot,ein denaturation represent a disruption of spatial order among chromophoric groups, a process which they liken to the loss of long range crystalline order upon melting and which is indeed a fitting description of a helix-coil transition. Cohen (1955a) was the first to propose that the native rotation of a protein results from the superposition of a specifically helical conformation upon an inherent rotatory background. Proceeding from the plausibility of helical structures in native proteins as suggested by X-ray evidence, she pointed out that the denatured forms of both globular pro-
498
PETER URNES AND PAUL DOTY
teins and collagen have similar specific rotations whereas the native forms of these two classes are respectively characterized by rotations in opposite directions from the common reference state. As became clear in subsequent studies upon synthetic polypeptides, the disruption of a standard polypeptide helix in fact produces increased levoro-80
-70
-
-60
-
I
I
I
I
I
I 40
I 60
80
[=I589
-50
-
-40 -
20
I
FIG.13. The thermal transition of insulin at pH 3.6 from the native to the denatured form, as followed by optical rotation. (Schellman, 1958c.)
tation, and this can be brought about by the same agencies that denature proteins, that is, pH, temperature, and hydrogen-bonding solvents. This is clearly shown in the pH and thermally induced transitions of poly-Lglutamic acid (Doty et al., 1957; Idelson and Blout, 1958), the pH dependence of conformation in copolymers of L-lysine and L-glutamic acid (Doty et al., 1958, Blout and Idelson, 1958), and the disordering effect of 8 M urea upon poly-L-lysine (Klemperer and Doty, 1961) and equimolar
OPTICAL ROTATION A N D PROTEIN CONFORMATION
499
copoly-L-lysine-L-glutamic acid (Doty el al., 1958). Thus the levorotatory change in specific rotation that accompanies the unfolding of a globular protein is compatible with its possession of helical regions in the native state.
5. Degree of Folding in Globular Proteins and the Rotatory Dispersion of Helices A correlation between the magnitude of the change in rotatory power upon denaturation, the degree of folding in globular proteins and, most importantly, their rotatory dispersion, has been ascertained by Schellman in a series of systematic investigations (Schellman, 1958b-f). In a preliminary communication, Linderstrplm-Lang and Schellman ( 1954) first suggested that a A, value above 240 mp for the simple dispersion of a native protein indicated the presence of a folded structure, while values less than 230 mp characterized disordered chains. Schellman then elaborated this initial correspondence by showing that proteins with the greatest change in levorotation, such as p-lactoglobulin, ovalbumin, and insulin, had high A, values, about 260 mp, as compared to the denatured state and that a number of intermediate cases on the two scales of A, and [ a ]were ~ provided by bovine serum albumin, chymotrypsin, chymotrypsinogen, and ribonuclease. Several proteins and protein derivatives known to lack secondary structure, clupein, the A chain of insulin, and oxidized ribonuclease had rotatory attributes indistinguishable from denatured, random chains. The amount of folded structure in these proteins as estimated thermodynamically by the heats and entropies of denaturation as well as from the temperature coefficient of rotatory power was reflected in the magnitude of A, and [a]689 for the same process of unfolding. On the hypothesis that this covariance represents the presence of a common folded structure, Schellman (1958f) proposed a scale for the mean residue rotation at 589 mp set by B-lactoglobulin, which undergoes a change in rotation, uncorrected for refractive index, of 93", and which was assumed to be completely folded. On this basis, the degree of folding in other globular proteins could readily be calculated, as shown in Table XI. (Schellman and Schellman, 1958). In the light of Schellman's suggestion that the dispersion parameters of proteins intermediate in folded content can be reproduced by appropriate mixtures of a completely folded protein and a disorganized chain (Schellman, 1958f), the parallel of this analysis to that for conformational mixtures of synthetic polypeptides becomes clear. Both schemes take their origin in dispersion parameters for two standard reference conformations, one ordered and the other disordered, and then adopt the conception that intermediate degrees of order can be gauged by the changes that ac-
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PETER URNES A N D PAUL DOTY
company its disruption, either by denaturation or a helix-coil transition.
All one need do is substitute the word “helical” for “folded” to see the essential similarity. The patterns of analysis are identical with respect to the behavior of rotatory power at one wavelength but differ in their use of rotatory dispersion, for one involves only simple dispersion with a change in A, whereas the other depends upon the parameters of mixed complex and simple dispersion without a change in dispersion constant. It is at, this juncture that the relevance of the complex dispersion of polypeptide helices to the simple dkpersion of native proteins may he made clear. TABLE XI The Degree of Folding of Native Proteins Estimated from the ChangP i n Optical Rotation with Denaturation’s t Protein fl-Lactoglobulin Ovalbumin Insulin Bovine serum albumin Chymotrypsin ?-Globulin Lysozyme Chymotrypsinogen Ribonuclease Pepsin
1 .oo 0.79 0.66 0.56
0.47 0.47 0.46 0.39 0.39 0.24
* (Schellman and Schellman, 1958.) t The change in mean residue rotation a t
589 mfi for fl-lactoglobulin in aqueous Rolution, 93”, is assumed to represent the loss of a completely folded structure.
At the same time that Schellman’s studies were in progress, an investigation of the rotatory dispersion of globular proteins in the light of synthetic polypeptide structure was being carried out (Yang arid Doty, 19.57). The rotatory dispersion of a poly-L-glutamic acid helix may be added in varying proportions to that characteristic of its disordered chain and the sum a t each wavelength, given by Eq. (30), then plotted according t o the simple Drude equation. As shown in Yig. 14, if the wavelength range is 350 t o 700 mpl linear plots are obtained up to about 40% helical content and A, values progress from that for the random coil, 212 mp, t o 268 mp for 4.0% helix while in the same interval the specific rotation, [aIM9, increases from -77 to -46”. Thus treating mixed synthetic polypeptide dispersion as one would a protein dispersion reproduces the same covariance of A, optical rotation, and degree of folding t,hat is observed in proteins and suggests t,hat, it is the explicitly helical nature of t,he fold-
50 1
OPTICAL ROTATION AND PROTEIN CONFORMATION
ing that gives rise to the changes in dispersive behavior. This correspondence implies that we can equally well treat protein dispersions as one would a partially helical standard polypeptide like poly-L-lysine a t stages in its helix-coil transition and thus use one suitably calibrated equation of Moffitt form with a single variable, helical content,, to express the dispersion data for all globular proteins for which A, is greater than that for their denatured forms. The fact that the same dispersion data will satisfy both simple Drude mid complex Moffitt behavior is, as has been shown in connection with
I
-300
I
I
-200
I
I
[a] -100
f
0
FIG.14. Hypothetical mixtures of dispersion data for the helical and randomly coiled forms of poly-L-glutamic acid according t o the simple Drude equation, together with experimental points for the two pure conformations, as shown previously in Figs. 1 and 2. The curvature characteristic of complex helical dispersion clearly emerges only as the proportion of helix increases above 40%. The dashed line indicates points corresponding t o 589 mp. (Yang and Doty, 1957.)
pseudocomplexity (see Section 111, C , I ) , a function both of the Wavelength range in which the data are obtained and the ratio of bo to uo'. So long as the wavelength range is above 350 mp, a ratio between -0.2 and +0.6 will yield linear plots of Eq. (27). The coefficients uo and bo are usually both negative, so that as bo decreases, A, must increase, thus showing that both bo and X, can be used as a measure of helical content. That A, in fact reflects helical content is shown by its rise in proteins for which there is independent evidence that helical content is being increased. The nonaqueous transitions of insulin, native and oxidized ribonuclease, silk fibroin, and oxidized bovine serum albumin (Yang and Doty, 1957) as well as that for carboxymethylated human serum albumin (Bresler
502
PETER URNES AND PAUL DOTY
el al., 1959) all show increases in A, to well above native values. Thus in this context there is good reason to think that intermediate values characteristic of native proteins in aqueous solution are on the same spectrum of helical content, a spectrum that can be revealed by a combination of urea denaturation to one side of the native state and what is essentially a reverse denaturation by 2-chloroethanol, dioxane, or dimethyl formamide to the other. This analysis imputes to native protein dispersions, like those of partially helical poly-L-lysine, some inherent complexity that is elicited by the Moffitt form with A. set to 212 mp, and carries the implication that those dispersions with a large proportion of complexity should deviate from linear Drude behavior a t higher wavelengths than those with low inherent complexity. Schellman and Schellman (1956) observed that a Drude plot for bovine serum albumin deviated progressively from linearity below 400 m p , while that for ribonuclease remained linear down to 300 mp. Jirgensons (1961c), in amplifying this observation by dispersion measurements on a variety of proteins between 296 and 365 mp, has concluded that proteins which have A, values above 250 mp, such as human serum albumin, ovalbumin, and lysozyme, show mildly complex deviations that increase as the wavelength is lowered, a behavior strikingly reminiscent of the calculated Drude dispersion for 40 and 50 % helical content in poly-L-glutamic acid (Fig. 14). I n contrast, those with A, values below 250 mp exemplified by B-lactoglobulin, a-chymotrypsin, and ribonuclease continue to exhibit simple dispersion. This result is thus in accord with an inherent complexity in the simple dispersion curves above 365 mp that is reflected in both A, and bo . I n some of the first protein dispersions measured, Hewitt (1927) took the coincidence of A, for a variety of serum albumins, ovalbumin, and lactalbumin with the tyrosyl absorption band a t 276 mp to mean that tyrosine was the dominant chromophore controlling these dispersions, a correlation which he reinforced by pointing to the low A, of gelatin, which has little tyrosine. Quite apart from the objections to this manner of interpreting the dispersion constant, synthetic polypeptides transparent above 250 mp demonstrate that no side-chain absorption or chromophoric shift need be associated with A,. Murakami (1957), in a proposal essentially similar to the treatment of protein dispersions by Moffitt form, has indicated how complex dispersion of inverse square dependence upon (Az - A;) will lead to high A, values if A0 is set to 190 mp to coincide with t,he longest wavelength peptide absorption band. It is also worth recalling here the formal description of the behavior of A, in the initial stages of the onset of complex dispersion such as that which accompanies the formation of a helix from a randomly coiled polypeptide (see Section 11,D). In terms
OPTICAL ROTATION AND PROTEIN CONFORMATION
503
of the phenomenological definition of A,, Eq. (21), it can increase as a result of a rise in rotatory strength of an optically active transition a t relatively high wavelength as well as from a spectral shift. The same effect can be achieved by gradually mixing a helix characterized by a strong rotatory contribution and Cotton effect near 225 mp (Fig. 4) with a random coil which lacks this power. It is important to emphasize that the rotatory properties which differentiate a native protein from its denatured form are a distinct consequence of its conformation rather than chemical changes inherent in the denaturing process. Denaturation rarely alters the organic composition of a protein, while, as already mentioned, the available data indicate that pH, urea, and temperature, all common denaturing agents, have relatively little influence of their own upon the A, and [a]689 values for protein chains (Schellman, 1958e). Of the possible conformations, it is clear that a chain arrangement consisting of helical segments mixed to a varying degree with disordered regions can account for the salient features typical of native dispersions. To review briefly what these features are, one may arbitrarily divide rotatory dispersion into three spectral regions. In the visible spectrum, t,he simple dispersion of the native form with A, and [aIs89 values greater than those for the disordered chain has been thoroughly documented. As the ultraviolet region is included down to about 300 mp, a range that has been less well studied, the dispersion becomes complex for globular proteins with high A, values derived from visible measurements alone (Schellman and Schellman, 1956; Jirgensons, 1961~).And in the far ultraviolet spectrum, several fibrous proteins and the globular proteins myoglobin and hemoglobin have been shown to exhibit complex dispersion, marked levorotation, and a sharp minimum near 233 mp, features that disappear upon denaturation (Simmons et aE., 1961; Beychok and Blout, 1961; Urnes et al., 1961a). Seen as comprehendingall three regions of the spectrum, the rotatory dispersion of any given protein is a unitary phenomenon, one that can perhaps be described down to 240 mp by a single expression of Moffitt form, and, with the appropriate choice of A. , compared with other protein dispersions that adhere to the same equation. These, then, are the explicit properties that any conformation proposed as characteristic of native proteins must be able to reproduce. Neither the @-formnor the flexible, randomly coiled chain has these attributes. There remains the possibility that disordered chains in particular environments, either rigidly held or in media of low polarity such as might be expected at the interior of a globular protein, can give rise to these features, but they have been little studied by optical rotation. It therefore appears that the characteristic changes in optical rotatory dispersion which accompany the denaturation of many globular proteins can be explained by the disruption of helical regions which in the native
604
PETER URNES AND PAUL DOTY
state include up to 50 % or more of the polypeptide chain. One feature of denaturation, however, seems at variance with the relatively short helical segments that can fit within typical dimensions of globular proteins seen, for example, in the models of myoglobin and hemoglobin (Kendrew el al., 1960, 1961; Perutz et al., 1960). If unfolding in globular proteins is a helixcoil transition, then the transition is often surprisingly sharp (Fig. 13) in view of the broad transitions expected for short lengths of helix (see Section 111, H ; Fig. 11). Zimm et al. (1959) therefore propose that other cooperative effects in a protein molecule may considerably sharpen the transition of a short helix, and Scheraga et aE. (1961), have recently shown within the context of a theory of reversible protein denaturation (Scheraga, 1960) that cross-links and side-chain hydrogen bonds will in principle produce this effect. Copolymers of L-lysine and L-glutamic acid undergo sharp pH transitions that do not vary in pK with helical content (Blout andIdelson, 1958). Since these are random copolymers, one might expect helical regions in them to be short, so that the sharpness could result from glutamic acid side-chain interactions which may stabilize these helices (see Section 111, H ) . Taken together, these considerations hence suggest that the sharpness of denaturation does not exclude helix-coil transitions in globular proteins.
D. Scales of Helical Content as Applied to Proteins What accommodations, then, must be made in the pattern of analysis developed for standard synthetic polypeptides if it is to yield quantitative estimates of partial helical content in proteins? The requirements are much the same as those set out for poly-L-lysine (see Section 111, G, 3), yet since globular proteins, like copolymers of L-lysine and L-glutamic acid, cannot be made completely helical in aqueous solution, a helical reference conformation must be taken either from other standard molecules or from the nonaqueous behavior of the protein in question. This latter procedure involves solvent changes with little bearing on native conditions and may be impossible to carry out in the face of restraints imposed by proline and cross-links, so that standard helical dispersion can more feasibly serve as this reference conformation. A value of bf = -630 is well established for synthetic polypeptides and fibrous proteins. The considerations leading to the reasonable approximation that ba of the disordered chain is nero, together with the implied equality of Xo and A, for this state, have been discussed in Section 111, C, 1, and incorporated into the pattern of analysis for partial helical content as set out in Section 111, G, 2, A value of a I D = +650 has been obtained for poly-L-glutamic acid, poly-L-lysine, and Pinna nobilis tropomyosin under the appropriate conditions. As has been stressed, these constants have conformational significance only when a value of 212 mp is used for X o . A dispersion constant of 212 mp with its corresponding values of b f and
OPTICAL ROTATION AND PROTEIN CONFORMATION
505
can continue to serve for data above 300 mp, but another value of Xo may become necessary if further measurements upon water-soluble helical polypeptides and fibrous proteins corroborate the finding that a constant of 212 mp fails to give linear Moffitt plots at lower wavelengths. In a case already cited (Section 111, B ) , a value of 216 mp is required to deal with data down to 240 mp for a standard helical polypeptide, a copolymer of 5 % L-tyrosine with L-glutamic acid, and this value, together with a new b f of -535, has been used in the treatment of the ultraviolet dispersion of myoglobin (Section 111, 0). Helical poly-L-glutamic acid requires a similar change in ho to 218 mp, as previously described. Moffitt plots of the ultraviolet data from 240 to 280 mp for tropomyosin and paramyosin as reported be Simmons et al. (1961) yield bo values of about -900 when Xo equals 212 mp, as does the plot of copoly-L-tyrosyl-L-glutamic acid if it is confined to this narrow wavelength range (Fig. 8). With X o set to 216 mp, the same protein data give bo values of about -560. These calculations thus suggest that Moffitt plots covering the full dispersions for these helical fibrous proteins will exhibit curvature with Xo set to 212 mp, although a detailed examination of the data will be required to determine the specific value of ho that renders the plots linear. If A 0 is systematically changed in order to encompass the full spectral range of protein dispersions, then the reference values of br and a:-* must be altered accordingly for consistency. The standard value for the remaining constant, A[m']fif, may be chosen on a variety of bases. The values for typical water-soluble polypeptides, poly-L-glutamic acid, about 85", and poly-L-lysine, 95", together with that for Pinnu nobilis tropomyosin, 85", average to 88". If this is translated to the specific rotation in water for a mean residue weight of 115, A[a];i; is 97". An arbitrary choice may be made by setting A[m']gf to 90" and A[a]?i: to 100". Once a value for bib8 is obtained from the dispersion of a native protein, f H can be evaluated directly with Eq. (36), but the use of the other two scales requires the denatured state to serve as a reference conformation. Its value for [2']f89 will thus set the lower limit of A[m']fi:, and the intercept of its Moffitt plot will provide a% , both of which are necessary to computefH from Eqs. (34) and (35). In a manner indicated in Section 111,C, 1, a t may here be interpreted as the mean intrinsic residue coefficient, a,", for the various species of residue present. This coefficient often appears in Eq. (33) instead of the less familiar term u t ; if one realizes that the only direct measure of 6,"is a t , this interpretive substitution can on occasion lead to useful results, as will be seen in the work of Leonard and Foster (1961a,b; Section IV, G, 3). Since the scheme that has been developed to assess partial helical content is based on the approximation that X, of the disordered chain equals Xo , the measurement of X, will test the plausibility of this assumption in any given case. Values of [m']~embody a correction
506
PETER URNES AND PAUL DOTY
for refractive index, so that a value of n appropriate to the denaturing solvent should be used. It is, of course, essential that the denaturation be complete for these measures to be representative. Nonetheless, several considerations force us to recognize that the rotatory parameters of a denatured protein may fail to represent disordered regions in the native protein. For one matter, it is unlikely that nonhelical regions in a globular protein are truly random coils, for this would require a degree of flexibility incompatible with its compact structure. I n the crystalline state, furthermore, it appears that nonhelical portions are rigidly fixed, for Kendrew et al. (1961) are able to follow the chain direction and identify side chains in these regions of myoglobin at a 2 A level of resolution, a feat made possible only by the identical disposition of these portions in each molecule. These regions may be best described as amorphous or disordered and the relevance to them of rotations characteristic of a denatured protein or a random coil justified by their lack of periodic structure. According to the rule of Kauzmann and Eyring (Section IV, A ) , the b e d orientation of the chromophores may endow them in these circumstances with an enhanced rotatory power, but one may surmise that their dispersion would remain simple in the absence of an ordered conformation. Secondly, the rotations of the denatured form may not be typical of the amino acid composition actually present in shorter, amorphous sections of the native protein. The use of [m‘]& for reference purposes may thus include some change with denaturation that is attributable not to a loss of helical content but rather to a change in the species of residue. Moreover, the sensitivity of the rotation of a denatured protein to ionic strength, ionic species, and other solvent conditions, as has been demonstrated for randomly coiled synthetic polypeptides (see Section 111,C, 1 ), may introduce additional changes in the optical rotation that again give the polypeptide chain uncharacteristic rotations under denaturing conditions and further as scales of helical content. For interfere with the use of A[m’]fi-,” and these masons, bibs is a more reliable indication of helical content than either the change in optical rotation at one wavelength or the change in the intercept. A final feature of disordered regions, one which can affect the estimation of helical content from b;bs, is a difference between A, and A 0 . If A, for the simple Drude dispersion of the disordered regions is unequal to Xo , then a “pseudocomplex” contribution to bgbs will result (Section 111, C, 1). Estimates of partial helical content based on bibs and b f must therefore be understood to embody this range of uncertainty, one that becomes progressively greater as the proportion of chain in disordered array increases. For this reason, estimates of small helical content are especially unreliable. Even if deviations of A, from XO are taken into account by using Eq. (28) to calculate br and thereby subtract it from bibs, there is no assurance that,
OPTICAL ROTATION A N D PROTEIN CONFORMATION
507
a A, or bt value for the completely disordered chain will represent average values for the actual amorphous segments in the native structure. In the absence of any other procedure for dealing with these elusive regions, one can only employ the rotatory parameters of the denatured state with the hope that the various factors leading to unrepresentative values may to some extent cancel one another. Another measure of helical content recently proposed by Simmons et al. (1961) is the optical rotation at 233 mp, [~2’]2~3, a value which is to be compared with the sharp minimum observed at this wavelength in far ultraviolet dispersions of helical polypeptides and proteins (see Section 11, 0). This is essentially a measure of A[m’]f-”, in this case the difference between about - 13,000”for the completely helical form and about - 2,000” for the disordered chain, but it may have the advantages that the rotations at this wavelength primarily reflect transitions peculiar to the helical backbone rather than side chains and that it is a true minimum which can be definitely located. This parameter does appear to correlate well with bo as an index of helical content. It is, however, essential that solutions with absorbances below the limit at which stray light effects begin to appear be used for conformational analysis at 233 mp. As will be discussed (see Section IV, J), solutions of high absorbance can give rise to artifactual deflections in this spectral region that may be mistaken for the authentic Cotton effect features. As a safeguard against this deception, the dependence of optical rotation upon the amount of protein in the light path should be determined and dispersion measurements to locate the true minimum near 233 mp carried out. Since peptide absorption at 233 mp is rising rapidly, thus confining one to low rotations, it may be convenient to calibrate a scale at somewhat higher wavelengths as well. It is important to note that even in solutions of low absorbance a lack of spectral purity can affect the accuracy with which the amplitude of this Cotton effect feature is measured. Even though the monochromator may be set at the true minimum of a Cotton effect, the beam will contain light at wavelengths to either side of it and hence give rise to a net rotation somewhat more positive than tjhe true extreme value. The sharper the minimum, the more pronounced this error will become, so that the amplitude for completely helical structures will be more seriously underestimated than for partially helical molecules. The use of the amplitude of completely helical forms as a reference will thus tend to produce overestimates of partial helical content. There has been little systematic study of instrumental factors upon this type of measurement, so that the seriousness of this error is not known. In any case, one can say that the slit width should be as small as possible and identical for measuring both reference and unknown conformations. The majority of protein dispersions have been submitted only to Drude analysis, even though the difference in X, between the native and denatured
508
PETER URNES AND PAUL DOTY
state receives only occasional use as a quantitative measure of helical content (Yang and Doty, 1957). The corresponding Moffitt parameters may be computed in two ways, either by direct calculation of optical rotations a t typical wavelengths using the recorded Drude parameters of A, and [?dl689 and then replotting these as “data” for Moffitt treatment, a procedure used by Schellman and Schellman (1961) or by an equation linking Drude and Moffitt dispersion as employed by Tanford el al. (1960). If A, and [m’]68g values of a simple dispersion are known, the corresponding bo can be computed in this latter way if the dispersive expressions are equated a t two wavelengths to permit elimination of a. . The intercept a. is generally not reported for Moffitt plots, but it can be calculated if [m’]68g and bo are known, as Tanford el al. (1960) have done for a variety of proteins and polypeptides. The primary reservation concerning these procedures is that [m‘]689 values measured at the conventional high wavelengths may have been somewhat discounted in the initial drawing of slopes in both the Drude and Moffitt treatments, 50 that the parameters subsequently calculated from them may not be entirely representative of the actual dispersions (see Section 11, B and Section 111, B ) . A number of routine methodological observations may be made at this point. Absolute rotations can be measured with relatively great precision, often with an average error of =t0.002”,so that rotatory data accurate to within 1% may be obtained. The graphical assessment of the dispersion parameters, A, , bo , and a0 , becomes more precise as the wavelength range is increased, but in general these values are reproducible only to within several per cent. Jirgensons (1961a) estimates the error in A, to be f 2 or f3 mp and stresses that it may be larger. On the other hand, Leonard and Foster (1961b) have been able to reproduce bib’ to within f l % for single sets of dispersion data. The uncertainties inherent in these determinations are of course compounded as any one value is placed on a standard scale to obtain helical content. The use of rotatory dispersion for conformational analysis of a single protein such as has been carried out by Leonard and Foster (1961a,b) for bovine serum albumin and by Jirgensons (1961~)for human serum albumin may be relatively sensitive to small differences, but in view of imponderable factors in an extrapolation from one protein or polypeptide to another, the best estimates of helical content, by any one parameter can be considered valid only within = t 5 9%. Since all dispersion parameters are placed on a residue basis, it is essential for comparative purposes that the purity of the sample be high and concentrations measured accurately. The calculation of reduced molar rotations, [m’]* from the observed rotations, ax, can be based on the composition of the protein concerned or a mean residue weight of 115 assumed if this is not known. Further details of polarimetric techniques are available in a number of good instrumental accounts. A revision of Heller’s comprehensive review
509
OPTICAL ROTA'ITON AND PROTEIN CONFORMATION
has recently appeared (Heller and Fitts, 1960), and the characteristics of instruments in current use are discussed by Djerassi (1960) and by Klyne and Parker (1960). Todd's review (Todd, 1960) is devoted in part to methods appropriate to proteins and polypeptides.
E. Helical Content from Rotatory Dispersion and Other Methods The capacity of rotatory dispersion to assess partial helical content in synthetic polypeptides has been demonstrated both by its self-consistency and general agreement with hydrogen-deuterium exchange. If this same patkern of analysis discerns the presence of helical segments in globular TABLEXI1 Partial Helical Content of Native Proteins in Aqueous Solution Eslimaled from Optical Rotatory Dispersion*, t
Tropomyosin Insulin Bovine serum albumin Globin-M Ovalbumin Lysozyme Pepsin Histone Ribonuclease Globin-H
0.88 0.38 0.46 0.37 0.31 0.29 0.31 0.20 0.16 0.15
0.87 0.57 0.58 0.52 0.50 0.39 0.26 0.30 0.17 0.09
0.91 0.59 0.46 0.53 0.53 0.37 0.26 0.26 0.17 0.12
0.89 0.51 0.50 0.47 0.45 0.35 0.28 0.25 0.17 0.12
* (Doty, 1957; Imahori and Doty, 1957; Doty, 1958.)
t The constants beH =
-630, a F D = +650, and A[m']&-' = 90".
proteins, its results should satisfy these same criteria. Table XI1 lists the percentage of helix for a variety of native proteins as measured both by bf and (Doty, 1958, 1959) as well as data from an earlier discussion in which values based on slightly different constants were compared with [m'IM9 values (Doty, 1957; Imahori and Doty, 1957). This tabulation shows that the agreement among the parameters for any given protein is generally good to within 10% of the full range of helical content and that the parameters taken together reveal a definite spectrum of proteins in terms of this variable. Estimates of helical content for these same proteins in 2chloroethanol appear in Table XIII, which is presented in this context as further evidence for the self-consistency of the rotatory parameters. Although the results both for native proteins and those with increased helical content are not nearly as coherent as for poly-L-lysine, in view of the ap-
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PETER URNES AND PAUL DOTY
proximations that have been made, the reservations pertaining to the use , and complications yet to be discussed, the met,hod of A[m']?$ and displays a reasonable internal consistency. The estimates of Schellman and Schellman (1958) in Table XI for degree of folding based upon the mean residue rotation in water against a scale set by p-lactoglobulin, 93', are generally higher than those recorded in Table XII. Since there is now reason to suppose that the large change in levorotation upon denaturation of this protein may result from effects other than loss of helical content, its scale may be replaced by the one proposed above, in which case the full change in the mean residue rotation a t 589 mp, unTABLEXI11 Partial Helical Content oj Proteins i n I-Chloroethanol Solution Estimated from Optical Rolatory Dispersion+*f Protein
Tropomyosin Insulin Bovine serum albumin Globin-M Ovalbumin Lysosyme Pepsin Histone Ribonuclease Globin-H
1.08 0.38 0.62 0.81 0.75 0.47 0.32 0.67 0.62 0.71
1.14 0.54 0.91 0.86 0.98 0.81 0.58 0.79 0.76 0.82
1.14 0.53 0.85 0.85 0.94 0.79 0.57 0.77 0.72 0.79
1.12 0.45 0.79 0.83 0.89 0.69 0.49 0.74 0.70 0.77
* (Doty, 1957; Imahori and Doty, 1957; Doty, 1958.) t The constants boH = -630, = +650, and A[WL']&-~ = 90". corrected for refractive index to agree with these values, is 113'. For those proteins appearing in both tables there is then as good agreement as exists among the parameters of Table XII. Blout el al. (196l), using an infrared spectroscopic method to follow the rate of amide hydrogen-deuterium exchange, estimate the percentage of "hard-to-exchange amide hydrogen" for the proteins listed in Table XIV. Although they use a helical polypeptide, poly-L-glutamic acid, as a model to demonstrate the capacity of the method, they stress that other factors in addition to intramolecular hydrogen bonds can reduce the rate of exchange. I n all cases some residual fraction of the hydrogen has not exchanged a t 24 hr. Their estimates, however, compare reasonably well with those reported by LinderstrGm-Lang (1958). Proceeding from the initial observation of Imahori and Tanaka (1959)
511
OPTICAL ROTATION AND PROTEIN CONFORMATION
that poly-L-glutamic acid undergoes a considerable hypochromism at the 190 mp peptide absorption band upon forming a helix, Rosenheck and Doty (1961) have calibrated this effect in terms of helical content with TABLEXIV Hydrogen-Deuterium Exchange as a n Index of Partial Helical Content in Native Globular Proteins* Protein
Approximate pD
% HEAHt
Average deviation (%)
Insulin Ovalbumin Lysoz yme Chymotrypsin Bovine serum albumin Ribonuclease B-Lactoglobulin Chymotrypsinogen Trypsin 7-Globulin
2 5 4.5-5 4.5 5 4.5 5-5.5 4 4 4
60 50 45 43 35 35 25 20 14 <10
f4 f5 f7 f6 f6 f5 f6 f4
*2
-
~~~
* (Blout et al., 1961.)
t HEAH: hard-to-exchange amide hydrogen. TABLEXV Partial Helical Content of Proteins in Aqueous Solution Estimated f r o m Hypochromism of Peptide Bond Absorption Near 190 mp* Protein
190 mp
197 mp
205 mp
Average
Paramyosin Myoglobin Insulin Ribonuclease, Native Oxidized @-Lactoglobulin, pH 6.4 pH 8.7
1.05 0.99 0.96
0.93 0.67 0.44
1.02 0.81 0.57
1.00 0.82 0.66
0.58 0.18
0.35 0.18
0.27 0.19
0.40 0.18
0.33 0.23
0.25 0.14
0.31 0.12
0.30 0.16
* (Rosenheck and Doty, 1961.) synthetic polypeptides in order to apply it to proteins. A correction for sidechain absorption, which can be gauged from the spectra of free amino acids and simple peptides, is required to obtain the extinction coefficient of the peptide bond. The fraction of peptide bonds in helical conformation estimat,ed in this manner at three wavelengths are listed for a number of proteins in Table XV. Considerable discrepancy exists among the three wave-
512
PETER UItNES AND PAUL DOTY
lengths, but the relative position of proteins in each column is the same. Although still in the process of development, this method appears to have the advantage of distinguishing a helix from the 0-form, for the latter has an extinction coefficient greater than that of the random coil and with a somewhat different maximum. Since the hypochromism at 190 mp is a phenomenon peculiar to the helical conformation and a slow rate of hydrogen-deuterium exchange is compatible with it, these methods provide independent support both for the contention that these globular proteins contain helical segments and the ordering of individual proteins on a spectrum of helical content. Estimates of helical coiiteiit hy rottltory dispersion appear in general to be lower than those provided by either of the foregoing methods. Because of the compact structure of globular proteins, it is possible that nonhelical amide groups in hydrophobic regions or in the @-conformationlead to overestimates by hydrogen-deuterium exchange, while the mode of correcting for side-chain absorption a t 190 mp may introduce considerable uncertainty. Yet one might anticipate rotatory dispersion to give inherently low estimates of helical content because of end effects (see Section 111, G , 1) and the possibility that some helical segments are of opposite sense. Furthermore, as mentioned above, if the simple Drude behavior of amorphous regions as expressed in X, and uD is at variance with the incorporation of these constants as XO and u t into Moffitt form, then estimates deviating in either direction from the actual value must be expected, especially if the fraction of helix is small. The agreement among these three methods is nonetheless qualitatively good. Various lines of argument thus converge to the conclusioii that globular proteins can contain up to 70 or 80 % of their residues in a helical conformation that is almost certainly a right-handed a-helix. The proven existence of right-handed a-helices in crystalline myoglobin, the agreement of the rotatory dispersion of this protein in solution with helical content in the crystal, the established capacity of rotatory dispersion to assess helical content in synthetic polypeptides, the manner in which complex helical dispersion can account for the simple dispersive attributes of proteins as well as complex features at lower wavelengths, the use of nonaqueous solvents to augment the native parameters in the direction of increased helical content, the self-consistency of this mode of analysis when applied to proteins, together with independent evidence from hydrogen-deuterium exchange and far ultraviolet spectroscopy, collectively substantiate the conformational hypothesis implicit in the use of the Moffitt form and coefficients. As seen clearly in the formal development of Eq. (33), this hypothesis asserts that helical segments and disordered regions coexist within a polypeptide chain and thus give rise to a net rotatory dispersion that is the sum of complex and simple dispersions, each characteristic of pure con-
OPTICAL ROTATION A N D PROTEIN CONFORMATION
513
formations studied in synthetic polypeptides. Yet structural features and alternative conformations not comprehended by this model, as highlighted by its inability to account for individual exceptions, serve to qualify the simple picture it presents for protein structure. It is to a consideration of these problems that we now turn.
F. DisulJide Bonds and Prosthetic Groups A prominent feature of globular proteins not present in synthetic polypeptide models is the disulfide bridge which makes large negative contributions to their rotation by virtue of its asymmetric configuration (Schellman, 1960). The rotatory dispersion of cystine shows the beginning of a Cotton effect associated with the disulfide absorption band at 255 mp, so that this structure can perhaps contribute to the complexity of protein dispersion and make difficult its analysis below 270 mp. Upon cleavage of the disulfide bond to two cysteine molecules, which have ordinary amino acid dispersion, [ a ]becomes ~ much less levorotatory, from about -200" for cystine to alone as an index of values near zero for cysteine, so that the use of [a]m9 conformational change associated with disulfide cleavage can be misleading. This effect was first shown for proteins containing cystine by Turner et al. (1958), who were able to account for the decreased levorotation in formic acid of bovine serum albumin, ovalbumin, and edestin after disulfide oxidation in terms of their cystine content and its expected change in rotation, and this finding was confirmed for additional proteins in aqueous solution by Wurz and Haurowitz (1961). It was thus suggested that the decrease in levorotation of ribonuclease and bovine serum albumin in nonaqueous solvents (Yang and Doty, 1957) and of human serum albumin in detergent (Markus and Karush, 1957a) after disulfide cleavage has this origin rather than arising from an increase in helical content. There is, however, evidence that this interpretation does not account for all the effects of disulfide cleavage upon optical rotation. The breakage of disulfide bonds can in fact permit the development in nonaqueous solvents of greater helical content than is possible for native molecules as indicated both by their rotatory properties and viscosity (Bresler et al., 1959; Frenkel and Horn, 1960), a finding which suggests that these cross-links act as conformational restraints in the native state. Disulfide cleavage can also lead to increased levorotation in water, as found for ribonuclease by Harrington and Schellman (1956), trypsinogen and a-chymotrypsin by Pechhre et al. (1958), human serum albumin by Jirgensons and Ikenaka (1959), and bovine mercaptalbumin by Kay and Marsh (1959b), observations which suggest that a loss of secondary structure overbalances the decreased levorotation resulting from the cleavage and that disulfide restraints are indeed required to stabilize t8henative conformation . I n an attempt to resolve the issue presented by Turner et al. (1958);
514
PETER URNES AND PAUL DOTY
Jirgensons and Ikenaka (1959) have been able to separate by rotatory dispersion and viscosity studies the conformational change consequent upon the breakage of cross-links from the changes in rotatory power a t one wavelength. They find that oxidized human serum albumin has a X, of 221 mp and the viscosity behavior of a linear polyelectrolyte, an observation confirmed by Bresler et al. (1959) for this protein and by Stauff and Jaenicke (1961) for bovine serum albumin in which disulfide bridges were cleaved by both oxidation and reduction. Oxidized human serum albumin is considerably less levorotatory than the untreated protein in denaturing solvents, but its conformation is undoubtedly that of a denatured protein. Kolthoff et al. (1960) have been able to achieve an apparent reversal of this type of denaturation by reforming disulfide bridges in serum albumin with an effective decrease in viscosity toward native values. The investigation of Harrington and Schellman (1956) on native and oxidized ribonuclease by rotatory dispersion and hydrodynamic methods, t,ogether with the subsequent study of Harrington and Sela (1959) on both the oxidized and reduced enzyme by similar techniques, establishes that disulfide cleavage in this protein also leads to a completely disordered chain characterized by a value for of about -90" and a X, near 225 mp. Harrington and Sela were able to increase both parameters for this structure by concentrated solutions of lithium bromide, thus reproducing an effect previously found by Harrington and Schellman (1957) for this and other proteins together with an enhanced thermal stability. Harrington and Schellman interpret these results to indicate the formation of helical segments owing to a lowered activity of water, but Bigelow and Geschwind (1961)) in a reassessment of their work, have suggested that a variety of salts including lithium bromide can increase of oxidized ribonuclease without conformational change, a view that is in harmony with salt effects upon the rotatory power of disordered polypeptide chains (Section 111, C, 1). One cannot, however, as yet discard the possibility of increased folding, for Bigelow and Geschwind report neither A, nor viscosity data for oxidized ribonuclease in concentrated salts, while Stracher (1960) in fact finds a retarded deuterium exchange for it under these conditions. Moreover, the increase in viscosity noted for the native protein in lithium bromide and sodium iodide does not rule out the development of new helical segments after an initial unfolding of the molecule. In a study on reduced ribonuclease that is of consequence for our concepts of protein structure, White (1961) has employed rotatory dispersion as one of the criteria by which he has demonstrated that air oxidation regenerates the native enzyme. As intlerpreted by him and by Anfinsen (1961)) this precise reformation of four disulfide bridges from eight distant cysteine residues provides good evidence that the amino acid sequence of ribonuclease predisposes its disordered chain to assume a characteristic spatial arrangement.
OPTICAL ROTATION A N D PROTEIN CONFORMATION
515
It may be relevant to the initial observation of Markus and Karush (1957a), the interpretation of which was called into question by Turner et al., that a number of other studies indicate that detergent can in fact stabilize and increase helical content in proteins. Pechhre et a2. (1958) observe a decrease in the specific levorotation at 589 mp of 25-30" for S-sulfo derivatives of trypsinogen and a-chymotrypsinogen in detergent, and Stauff and Jaenicke (1961) find similar changes in bovine serum albumin deprived of disulfide bridges. Jirgensons (1959a) has shown that detergent stabilizes human serum albumin against thermal denaturation without significant changes in rotatory dispersion, and, in a recent study (Jirgensons, 196le), finds that detergent may increase the helical content of proteins (yglobulin, for example), as judged from A, values that rise from 200220 mp t o above 250 mp. It is clear from these studies that rotatory dispersion, especially if it is supplemented by hydrodynamic techniques, provides more accurate information on conformational changes than do monochromatic rotations. In any case, the changes in monochromatic rotation caused by disulfide cleavage can always be anticipated and perhaps in many cases eliminated by calculation, as Turner et al. (1958) and Wurz and Haurowitz (1961) have been able to do. If proteins contain optically active absorption bands at wavelengths higher than the peptide bands, it may then be difficult to use rotatory dispersion for conformational analysis. Prosthetic groups can, for example, completely obscure the usual rotatory properties of proteins. The heme group in myoglobin, hemoglobin, the cytochromes, peroxidases, and catalases has strong optically active absorption bands in the visible spectrum that preclude direct assessment of peptide bond dispersion. If the Cotton effects a t these bands can be evaluated, then their rotatory contribution can in principle be subtracted to yield the inherent dispersion of the backbone. Although this kind of measurement is complicated by the large extinction coefficients concerned and its susceptibility to artifact (see Section IV, J ) , Cotton effects have been detected a t the characteristic heme absorption bands in cytochrome c (Eichhorn and Cairns, 1958), in myoglobin and hemoglobin (Klemperer and Doty, 1958; Savitz and Doty, 1958; Eichhorn, 1961; Beychok and Blout, 196l), in catalase (Eichhorn, 1961), and in sickle cell hemoglobin (Murayama, 1961). Enzymatic cofactors, such as diphosphopyridine nucleotide and flavin adenine dinucleotide, which have absorption bands in the near ultraviolet and visible spectrum, may influence the rotatory dispersion of their proteins at a time when the conformation of greatest interest, that of the biochemically active enzyme, is present. The dehydrogenases studied by Jirgensons (1959~)are generally isolated free of their cofactors, although crystalline glyceraldehyde-3-phosphate dehydrogenase, the dispersion of which does not appear
516
PETEH URNES AND PAUL DOTY
unusual, may be associated with diphosphopyridine nucleotide. The rotutory dispersion of alcohol dehydrogenase has been measured both in the absence of this cofactor (Ulmer and Vallee, 1961; Ulmer et al., 1961) and its presence (Ulmer et al., 1961). These latter workers have focused upon the negative Cotton effects that develop at the characteristic ultraviolet absorption bands of reduced diphosphopyridine nucleotide and its analogues as they attach to the enzyme and which they interpret in terms of asymmetric binding at the active site. Although binding thus produces negative rotatory contributions at higher wavelengths, it would seem that the conformation of the protein remains essentially the same, for the Cotton effects appear to be superimposed upon the dispersion of the free enzyme. The prosthetic groups in flavoproteins are, like heme, more firmly bound to the protein and thus may be expected to affect their dispersion. Carbohydrate may also influence the rotatory properties of a protein as in a-glycoprotein, which contains 17% hexose (Jirgensons, 1958a), has a A, of 249 mp, and a relatively small specific levorotation at 589 mp of -22” (Jirgensons, 1957). Ovomucoid (Jirgensons et al., 1960) and the myeloma globulins (Jirgensons, 1961a) also contain substantial amounts of hexose. Amino acids with a second asymmetric center, threonine, isoleucine, and hydroxyproline, must also be taken into account, but in view of the low rotations of the first (Schellman, 1960) and the relatively infrequent occurrence of all three, they do not affect protein dispersions to any large extent.
G‘. Rotatory Deviations from the Helix-Coil Pattern The proteins about to be discussed exhibit, as a common feature of their rotatory dispersions, a departure from the covariance of the rotatory parameters characteristic for a simple mixture of helical and disordered regions. Because of differences in amino acid composition and the sensitivity of the denatured form to solvent conditions, some lack of correspondence between the optical rotation at a single wavelength, [ m ‘ ],~and its change on denaturation, A[m’]f”, with the other dispersive parameters, A,, bo , arid a o , may be expected, but the discrepancies in these cases are sufficiently unusual to require further explanation. One group of exceptional cases is comprised of proteins with native A, values less than 230 mp that either remain constant, or rise upon denaturation while the specific rotation decreases in typical fashion. This decrease is compatible with some helical content, but the low A, values are at, variance with this interpretation. As tt second variety of exception, p-lactoglobulin has an affinity with this first group in that its specific rotation decreases with denaturation. Although this decrease is large enough to signify high helical content, the native value of X, , 249 mp, decreases only a moderate
OPTICAL ROTATION AND PROTEIN CONFORMATION
517
amount during the same process and bo remains constant a t a value indicative of a very small helical content. Since both P-lactoglobulin and most of the proteins with A, less than 230 mp have compact', folded structures, chain arrangements other than helices of standard sense must be invoked to account for their atypical rotatory properties. Another type of rotatory discrepancy is that found in a recent study of conformational change in bovine serum albumin. Although the specific rotation a t 589 mp for this protein decreases in the usual way with full denaturation and the dispersive parameters also change in a direction that indicates high helical content (Tables XI, X I I ) , during one of the intermediate stages in its unfolding the specific rotation a t 313 mp actually increases, while the other parameters show that helical regions are being disrupted. A detailed investigation of the structural basis for this exceptional behavior has led in this instance to a refinement of rotatory dispersion that may enable it to yield information on tertiary as well as secondary structure. 1. Proteins with Low Native A, Values
Of the small number of proteins with native A, values in the range usually encountered for denatured molecules, 210-230 mp (Table VIII), a- and p-casein have [cY]689 values, -90 and -140°, respectively, that are compatible with completely disordered chains, yet they appear to have some rigid, asymmetrical structure in the light of viscosity measurements that indicate axial ratios of greater than 10 (McMeekin, 1954). The more detailed studies on pepsin show that it has a A, typical of a denatured protein, 216 mp, but a specific rotation, [cY]689 , of about -664' (Blumenfeld et al., 1960) that is characteristic of considerable helical content. It can be unfolded a t high pH with a drop in [a]rn9to about -88" and a slight rise in A, (Jirgensons, 1958b). Although Perlmann (1959) has found similar trends on thermal denaturation, neither guanidine hydrochloride nor urea appear to affect either the specific rotation or A, other than by enhancing autodigestion, and in guanidine hydrochloride the intrinsic viscosity of the molecule differs little from the native form. Concentrated lithium bromide, makes the specific rotation slightly less levorotatory but does not affect A,. Pepsin is hence a globular, crystallizable protein in which hydrogen bonds are not the principle source of its stability and, judging from A , , there is no helical content. The bo and a. values, however, do indicate a helical content of about 29% (Doty, 1958; Table XII), which can be increased to 44 % in 2-chloroethanol, as shown in Table XIII. Since bo should be negligible for simple dispersion with a A, as close to 212 mp as 216 mp, the source of this discrepancy may be of an experimental nature. Perlmann reports that 62 % of the residues in pepsin are nonpolar, so that the globular
518
PETER URNES AND PAUL DOTY
structure of this unusual protein, which also contains large amounts of serine and proline, may perhaps be attributable to hydrophobic forces. I t is hence of interest that the precursor of this enzyme, pepsinogen, displays typical rotatory behavior with denaturation by heat and urea (Perlmann, 1961). X, decreases from 249 to 215 mp and the specific rotation at 400 mp drops from -156 to -270" in 8 M urea, changes which indicate that the structure is stabilized by intramolecular hydrogen bonds and that it has a moderate helical content. Luteinizing hormone, a protein that is similar to pepsin in having a X, of a denatured protein and moderate levorotation, [ ( Y I ~=~ ~-go", loses its biological activity in denaturing solvents without affecting either of these values. It also is a globular protein with relatively large amounts of serine, proline, and threonine. The X, value for soybean trypsin inhibitor, a protein reported to be globular (Jirgensons et al., 1960), is likewise typical of the denatured form while the specific rotation is somewhat high. Its several varieties contain substantial proportions of serine, proline, aspartic acid, and cystine, so that the amino acid composition of pepsin, luteinizing hormone, and soybean trypsin inhibitor would set them aside from other proteins and perhaps can account for their unusual structural properties. A number of serum proteins studied by Jirgensons (1958a, c, 1960a) all exhibit abnormally low X, values, below 210 mp, that rise upon denaturation to the usual range while, like the majority of proteins, their specific rotation at the same time undergoes a considerable increase in levorotation (Table IX). y-Globulin fractions, serum macroglobulins, and two groups of abnormal protein associated with multiple myeloma, the myeloma globulins and the excreted Bence-Jones proteins, all have considerable proportions,of the hydroxyamino acids, serine and threonine. The y-, macro-, and myeloma globulins apparently dissociate into well-defined fragments at high pH as measured by sedimentation, a process that is accompanied by some rupture of primary peptide bonds (Jirgensons, 1961a). If these proteins consist of subunits, then they may be stabilized by the p-conformation, which on a priori grounds is the most stable form of interchain hydrogen bonding. The X, values below 230 mp could, in all of the foregoing cases, be reconciled with the relatively small native levorotations by the presence of some @-conformationcharacterized by positive rotations and a positive bo . Numerical assessment of Eq. (27) (Section 111,C , 1) shows that A: is linearly related to ratios of bo to a. that can be negative as well as positive and that this ratio can be as low as -0.2 for rotations in the visible and near ultraviolet spectrum. Thus a positive slope and a negative intercept furnished by simple mixtures of &form and random coil will lead to A, values lower than 212 mp. It is possible to have a more complex mixture of disordered regions
OPTICAL ROTATION AND PROTEIN CONFORMATION
519
with standard helices and the p-form in which both of the latter conformations contribute positively to the optical rotation and yet keep A, below 230 mp and the observed bo to a small magnitude by their opposite contributions to these parameters. Equation (39) (Section 111,I) for a mixture of random coil, standard helix, and @-formcan in principle assign the proportions of these three conformations, thus expanding the simple helix-coil model to include this latter structure, but in view of the uncertainty surrounding both the rotatory properties of the @-conformationand the use of the Moffitt intercept for conformational analysis, the results of this kind of calculation cannot as yet be given much weight. The amounts of helix and p-form that would be present in proteins with X, less than 230 mp are probably not in any case large, for the native rotations are not sufficiently positive. The proposal that these proteins contain regions of p-structure can be independently tested by the characteristic infrared absorption frequencies of this conformation. Winkler and Doty (1961) have, for example, examined rabbit 7-globulin fractions by this means in connection with their rotatory and immunological properties and failed to find evidence of this structure. Schellman and Schellman ( 1961) have computed the rotatory parameters for mixed pairs of conformations based upon the coefficients characteristic of a typical random coil, oxidized ribonuclease; helices of opposite sense, poly-y-benzyl-L-glutamate and poly-P-benzyl-L-aspartate;and the differing @-formsobserved by Fasman and Blout (1960) and by Wada et al. (1961). Their tabulations clearly illustrate how A, decreases as the amount of @-conformation characterized by the Wada constants is mixed in increasing proportions with a random coil. Thus at 15% @-form,A, is 187 mp and [ c r ] ~ , has become 30" less levorotatory. A poly-P-benzyl-L-aspartate helix mixed with a random coil will also lead to low A, values, but in this case the optical rotations will progressively decrease. Aside from poly-P-benzyl-L-aspartate and standard polypeptides composed of D-residues (Table 111), the only evidence that is at all indicative of the existence of helices of this sense is the behavior of insulin in 2-chloroethanol.Unlike other proteins, its rotatory parameters in this solvent do not differ greatly from native values, as is shown in Tables XI1 and XIII. Lindley and Rollett (1955) have proposed a model for insulin in which about 20% of the residues are in left-handed helices as a result of restraints imposed by disulfide bridges. If one assumes that insulin does in fact become more helical in 2-chloroethanol, an initial mixture of right and left-handed helices will lead to no net increase in rotatory power as their segments are equally enlarged. Deuterium exchange and far ultraviolet hypochromism, which are capable of assessing total helix content instead of the excess of one sense over the other, do indicate higher values for insulin than does rotatory dispersion (Tables XIV and
520
1’ETES UItNES AND PAUL UOTY
XV) , but this evidence is probably not definitive since a similar discrepancy exists for other proteins as well. Although the interactions of L-amino acid side chains with a right-handed helix will be different from those with a left-handed helix, rotatory dispersion is primarily a function of the helical skeleton rather than side-chain interactions and cannot a t present distinguish among helices of different sense mixed with disordered regions. 2.
p- Lactoglobulin
An alternative explanation for small native levorotations accompanied by unusually low X, values that does not invoke the /3-conformation has arisen from a thorough investigation of the denaturation of p-lactoglobulin. As has been mentioned, all t,hree rotatory properties, [ m ’ ] ~ , A, , an.d 60 , for this protein are a t variance with one another in terms of a simple scheme of standard helices and disordered regions. The specific rotation a t 589 mp changes from -28 to - 117”with denaturation by urea (Schellman, 1958b), and on this ground the helical content would appear to be high. Indeed, Schellman originally chose /3-lactoglobulin as a paradigm for a completely folded protein. The native value for A,, 249 mp, changes during the same process of denaturation to 225 mp, an alteration that is compatible with only a moderate helical content. Yet bo for Schellman’s data, as plotted by Tanford et al. (1960) is only -68, an unexpectedly low value which they substantiate under a variety of conditions and which does not change appreciably upon denaturation at high pH. Tanford et al. interpret the low bo in a straightforward manner as indicating a negligible helical content in the native protein. They then suggest that the large increase in levorotation with unfolding can be explained by a solventlike effect upon the rotatory power of the peptide bonds as they leave a medium of low polarity a t the interior of the protein and enter an aqueous environment of high polarity. They adduce as evidence for the existence of a nonpolar interior in this molecule the hydrophobic stabilization that water appears to provide. As described in connection with the behavior of p-lactoglobulin in mixtures of water and nonpolar solvents (see Section IV, C , 3 ) , the levorotation initially increases with moderate proportions of dimethyl formamide or dioxane, a finding which is compatible with the unfolding of the molecule as hydrophobic forces cease to promote a clustering of nonpolar side chains a t its center. The Moffitt intercept, a0 , also becomes more negative as the protein is gradually denatured in these mixtures. The intercept for native protein is relatively positive, -169 to - 159, as compared to that for the denatured state in urea, - 663. If is set at +650, then this measure indicates a helical content of about 75%, a value obviously a t variance with that from b o aThe change in intercept, on thit interpretation, presumably reflects the solvent effect upon the mean
OPTICAL ROTATION A N D PROTEIN CONFORMATION
52 1
intrinsic residue coefficient, 6; , so that this coefficient cannot be taken as a constant over the process of denaturation, thereby preventing the use of the a r D scale. Solvent effects ran also be invoked to account for the change in X, , for X, of random coils is sensitive to solvent as shown, for example, by its drop from 210 mp for poly-y-benzyl-L-glutamate in hydrazine to 180 mp in dichloroacetic acid (Yang and Doty, 1957; see Section 111, C , 1). The fact that a moderately high value of X, for the native protein is compatible with a low bo is formally explicable by the small magnitude of the intercept, a. , for the native protein. In the context of Eq. (27), these values yield a bolaoratio of about 0.4, which is relatively large and hence leads to an equivalent value for X, that is appreciably greater than the value of Xo , 212 mp. It is possible, however, to make an alternative interpretation for the atypical variation of the rotatory parameters of p-lactoglobin in terms of mixed helical and @-content. If these conformations are present in about equal amounts, bo will be a small number, yet because the contribution of both conformations to [m']689 and a0 is positive, these parameters will decrease upon unfolding with little net change in bo , thus producing the same behavior as observed by Tanford et al. For what it is worth, a calculation of the fraction of helix and @-formcan be made with Eq. (39). If one uses the standard helical constants, the Wada constants for the @-form,the parameters observed by Tanford et al., and takes a: from the denatured protein as -663, then fH is 0.55 and fs is 0.33. Several pieces of independent evidence are compatible with the presence of helical regions and perhaps some @-structurein p-lactoglobulin. During its reversible transformation a t pH 7.5 (Tanford el al., 1959; Tanford and Taggart, 1961 ), a process apparently initiated without decreasing hydrophobic forces, the observed intercept drops from -169 to about -300 with an increase in levorotation of about 22" a t 589 mp and little change in bo (Tanford et al., 1960). Since far ultraviolet spectra suggest some loss of helical content in this transformation (Rosenheck and Doty, 1961; Table XV), the change in the rotatory parameters may in this case signify the partial disruption of both helical and @-regions.I n a sequel to the earlier paper, Tanford and De (1961 ) show that urea and formamide lower the specific rotation and unfold the protein in the same manner as low concentrations of dioxane, dimethyl formamide, and ethanol. Quite apart from the identity of the native conformation, it thus appears that both hydrophobic forces and hydrogen bonds contribute to the stability of P-lactoglobulin, although urea may weaken hydrophobic bonds as well (Kauzmann, 1959). The hydrogen bonds need not be periodically disposed, especially if there are other sources of stability, yet helices and @-structures remain plausible conformations for the optimal satisfaction of intra- and interchain hydrogen bonds, and if present, will influence the rotatory prop-
522
PETER URNES AND PAUL DOTY
erties. @-Lactoglobulin can be tested for the presence of @-structures by examination of its infrared spectrum. If measurements are carried out in D20, the analysis may, however, be somewhat complicated by the failure of amide hydrogens at the interior of the molecule to exchange, so that the band near 1610 em-’ characteristic of the deuterated 0-form may not appear and the customary band near 1630 cm-’ for the hydrogen-containing structure may be to some degree obscured by other conformations. The unusual rotatory properties of another protein, glutamic acid dehydrogenase (Jirgensons, 1961f), may be described in this context, for it is possible to interpret them in terms of mixed helical and @-content. As the protein is gradually denatured at alkaline pH, the specific rotation a t 546 mp decreases from +13 to -22” at 30 min and to -49” after 4 h r (Table VII). The value of bo , however, at first, decreases from - 170 in the native state to -357 a t 30 niin and then rises finally to -70. The initial changes in which [rw]b46 decreases while bo becomes significantly more negative are precisely the phenomena one would expect from the disruption of the @-aggregatesand the persistence of helical segments. Since this enzyme consists of subunits, it is conceivable that the @-conformationcould play a role in stabilizing the native structure. Two dist,inct explanations are therefore possible for the rotatory discrepancies observed in p-lactoglobulin and globular proteins with X, values less than 230 mfi. The one accounts for the relatively positive native rotations by solvent-like effects upon an essentially disordered chain, an arrangement that gives rise to a dispersion in large part typical of randomly oriented peptide bonds. The other brings the deviant rotatory parameters back into correspondence by introducing an additional ordered conformation for the chain, the @-structure,one that has dispersion characteristics capable of reconciling the observations. Neither alternative is, however, well grounded in the properties of model compounds. There is little evidence to indicate that a nonpolar environment will induce specifically positive shifts in the rotation of a disordered polypeptide chain, and although considerable progress has been made in characterizing the 0-form by rotatory dispersion, further study, especially in aqueous solution with higher molecular weight polypeptides, is required before this conformation may be attributed to native proteins. 3. Bovine Serum Albumin
I n the course of investigating conformational alterations in bovine serum albumin, Leonard and Foster (1961a) have closely analyzed changes in rotatory dispersion that are not paralleled by the specific rotation. As the p H is lowered, bovine serum albumin undergoes an “isomerization” detectable by two different electrophoretic components before it expands a t low pH.
OPTICAL ROTATION A N D PROTEIN CONFORMATION
523
These authors have found that the decrease in specific rotation a t low p H is preceded by a moderate increase, measured a t 313 mp, which is coincident with the isomerizing process as followed electrophoretically. The selective action of thiocyanate and perchlorate has enabled them to resolve this process into two well-defined optical transitions, one of which coincides with electrophoretic change and the other with the appearance of a solventinduced difference spectrum for tyrosyl residues. One might expect the increase in specific rotation to reflect an increase in helical content, but dispersion data a t various pH's in the isomerization indicate that the opposite occurs, for the helical cont,ent by bo drops from 48 to 40 %. At first glance, it would appear that the isomerization superimposes a positive rotation upon the increasingly negative rotations that should accompany the loss of helical content. However, in order to locate the source of this discrepancy systematically, Leonard and Foster propose two tests for the invariance of the helical parameters, a:-D and bf, and the mean intrinsic residue coefficient, &, over a set of different dispersion measurements. As has been discussed (Sections 111,C , 1 and IV, D),a? is a constant that interprets the Moffitt intercept for the disordered form, u;, as a reflection of the mean intrinsic residue rotation of the protein. It may be substituted for u t in Eq. (33) and the following analysis performed on the resulting expression. One test for the constancy of the coefficients is to plot the ratio of each intercept, dbs, to its slope, b$bs, against the reciprocal of the slope, a procedure which defines a new function,
in which 6," can be derived from the slope and the ratio a;-"/# from the intercept. If the coefficients do not vary from dispersion to dispersion, then this plot will be linear. Should there be reason, however, to think that 6,"alone changes over the transition, its variability will become apparent if it is first calculated as a function of a;bs and b:bal
and then plotted against bibs, which is here taken as a n index of helical content. From this type of analysis, Leonard and Foster conclude that a change in the mean intrinsic residue coefficient best accounts for the lack of correlation between the monochromatic rotations and the dispersive parameters, and they tentatively ascribe this change to alterations in tert,inry structure which might affect vicinal interactions, side-chain hydrogen bonds, and so
524
PETER UItNKS AND PAUL DOTY
forth. They arc thus able to estimate that the observed increase of about 30", which is 5 % of the total rotation a t 313 mp before isomerisation, represents a negative contribution of 60" due to a loss of about 8 % helical content masked by a positive contribution of 90" due to the change in tit. They point out that the specific rotation will be most sensitive to alterations other than helix disruption near the wavelength at which the helix and coil have equal rotations. Both by their computation and measurement upon a copolymer of 5 % L-tyrosine with L-glutamic acid (Urnes et al., 196lb), this crossover point is found to be near 295 mp (Fig. 9). This sensitivity can perhaps illuminate an observation of Perlmann (1961 ), who, by following the specific rotation a t 366 mp, has found a t,wo-step transition in the thermal denaturation of pepsinogen. The large change near 50°C and the smaller one near 65°C may hence represent stages that distinguish the disruption of helical regions from other structural changes. A lack of similar sensitivity at higher wavelengths is illustrated by the fact that no change in rotation occurs a t 589 mp in the isomerization of bovine serum albumin, so that the partial loss of helical content and increase in rotation a t 313 mp that characterize this process apparently compensate each other exactly a t this wavelength. One additional advantage of the treatment of Leonard and Foster is that the use of Eq. (40)also permits one to assign a r Dand d t for a protein in the absence of the completely helical and denatured forms, so long as one assumes a value for bf, the transitional range covered is not narrow and a sufficient number of dispersions are measured. For this procedure to be feasible, at, which is given by the slope of Eq. (40), must remain effectively constant over the transition. In a preliminary communication, Leonard and Foster (1961b) report that experiments similar to those described above but carried out a t constant p H (6.8) with increasing concentrations of detergent instead of increasing acidity, give evidence of a cooperative binding that closely follows the sequence of isomerization. I n a study that is relevant to the isomerization process in bovine serum albumin, Kauzmann (1958) has shown that volume changes in the protein near pH 4 are not paralleled by the optical rotation. He finds that the partial specific volume of this protein in water decreases below pH 4 concurrently with both the increase in viscosity and decrease in specific rotation that mark the well-studied reversible expansion, and, on analogy with like contractions in urea, suggest,s that the expansion is an unfolding similar to denaturation. The addition of 0.15 M potassium chloride suppresses the p H a t which rotatory arid viscosity changes occur without, however, a comparable quenching of the volume change. Kausmann's conclusion that optical rotation and volume change probably reflect different aspects of protein structure is considerably sharpened by the work of Leonard and Foster.
OPTICAL HOTATION AND PROTEIN CONFORMATION
525
They confirm that 0.1 2cI potassium chloride lowers the pH a t which the expansion occurs and yet show that it does not greatly affect the isomerization near pH 4. Since the isomerization is undetectable a t 589 mp, the wavelength used by Kauzmann, the optical rotation at this point will not parallel changes as indicated by other techniques. The volume contraction which continues in 0.15 M potassium chloride near pH 4 may therefore reflect a slight unfolding during isomerization, a possibility that is in harmony both with the concomitant, exposure of about 20% of the tyrosyl residues in bovine serum albumin as measured b y difference spectroscopy and the small loss in helical content. The resolution of the atypical behavior of the rotatory parameters during the isomerization of bovine serum albumin has therefore shown that rotatory dispersion can illuminate structural alterations more complex than a helix-coil transition. I n the usual assignment of conformation by rotatory dispersion, it is assumed that 6remains constant, so that small alterations in the rotatory power of individual residues are neglected. Indeed, for a molecule as simple as poly-L-lysine, the consistency of the standard pattern of analysis a t stages in its helix-coil transition provides good evidence that a t for its single species of residue in fact remains constant. It is, however, small alterations of this sort that one would expect to accompany changes in an intricate tertiary structure of a protein affecting the environment and orientation of diverse side chains. These changes would normally not appear in the customary measurements upon only the native and denatured forms, but the detailed treatment by Leonard and Foster of changes in the dispersion during the course of the isomerieation, together with optical rotation measurements a t a wavelength relat>ively insensitive to secondary structure, has in fact yielded information concerning the intrinsic rotatory power of the residues, information that can be correlated with other techniques. Although rotatory dispersion remains primarily a function cf secondary structure, its deviations from the consistent rotatory behavior of synthetic polypeptides may, upon systematic analysis, suggest ways in which proteins differ from their less complicated analogues.
H . Optical Rotation and Studies of Tertiary Structure The denaturation of fl-lactoglobulin by nonpolar agents and the conformational effects of disulfide cleavage upon serum albumin and ribonuclease demonstrate that sources of stability in addition to helical segments may be required for the rigid compact shape of globular proteins. A number of further studies upon the respective contributioiis of secondary and tertiary structure to this stability may be selected to illustrate the role that optical rotation, used as a method solely for determining secondary structure, can play in conjunction with other methods of structural assessment.
526
PETER URNES AND PAUL DOTY
Markus and Karush (1957b) find that X, of human serum albumin, 266 mp, is the same in water as in 8 % detergent and that [a1689is constant over much of the range of detergent concentration, although at low concentration the specific rotation increases slightly, a n effect which they attribute to ionization of e-ammonium groups and consequent conformational change. Yet the viscosity and availability of disulfide bonds for cleavage increases with detergent concentration, a finding which suggests that detergent separates helical segments without disrupting them. An analogous effect has been observed by Weber and Tanford (1959) for aqueous solutions of ribonuclease as the proportion of 2-chloroethanol increases. As the nonpolar component is initially added, they find that the specific rotation increases slightly and bo decreases from -95 to - 175, while the viscosity increases markedly with a n ionic strength dependence of a flexible polyelectrolyte. They interpret this behavior as the loosening of a compact molecule by a reduction in hydrophobic forces without significant alteration of the moderately small native helical content. Evidence that secondary as well as tertiary structure can be stabilized by hydrophobic forces is provided by the thermal denaturation of ovalbumin in aqueous mixtures of nonpolar solvents. Bresler et al. (1959) find that the temperature a t which the optical rotation of ovalbumin undergoes a transition in mildly alkaline solution is remarkably lowered in 30% dioxane and 4 M dimethyl formamide. Since the transition indicates the loss of helical content, it appears that the hydrogen-bonded structure of water in this case promotes helical stability. Spectrophotometric techniques as well as hydrodynamic methods have been employed to supplement information from rotatory dispersion. Harrington and Schellman (1956), in a comparison of native ribonuclease with the protein denatured in 8 M urea, find that the tyrosyl absorption band centered a t 277.5 mp shifts to 275 mp as the molecule unfolds, a process shown to occur by characteristic changes in viscosity, sedimentation, and rotatory dispersion. The enzymatic activity of ribonuclease nonetheless persists in 8 M urea (Anfinsen, 1956), an observation which challenges the general view that a protein requires some degree of secondary and tertiary structure in order to function. By demonstrating that analogues of the substrate such as uridylic acid, polymetaphosphate, and other polyvalent anions induce a partial refolding of the molecule in this solvent as judged from a reversal of the spectral shift, the viscosity and rotatory dispersion toward native values, Sela el aE. (1957) turn this surprising finding into support for the generalization. Foss ( 1961) has used difference spectroscopy to follow spectral shifts in a side-chain chromophore, either tyrosine or tryptophan, as the temperature is raised, and has thereby observed transitions in ribonuclease, lysozyme, and chymotrypsin that implicate these residues in the unfolding process. Since the spectral shifts are to lower wave-
OPTICAL ROTATION AND PROTEIN CONFORMATION
527
lengths, as is characteristic of denaturation, and since they can be produced by transfer of these chromophores to more polar environments, this phenomenon is compatible with the existence of nonpolar domains in a native protein and hence with stabilization by hydrophobic forces. The spectrophotometric transitions observed by Foss coincide with polarimetric transitions except for lysozyme at pH 2.2, for which the spectral shift of tryptophan occurs at 55°C while the decrease in specific rotation occurs at 80°C. The separation of two discrete transitions thus implies that the environment of tryptophan side chains changes before helical breakdown takes place, so that the denaturation of lysozyme may involve the sequential disruption of tertiary and then secondary structure. Dioxane and urea each depress both types of thermal transition, a finding which Foss interprets as evidence for the mutual stabilization of the native protein by hydrogen bonding and hydrophobic forces. A chromophoric shift to lower wavelengths may, however, result from the disruption of secondary as well as tertiary structure, for t,yrosyl residues copolymerized with glutamic acid display a similar shift in the first stages of a transition from helix to random coil (Urnes et al., 1961b). Spectrophotometric titration of tyrosyl residues has also been followed in conjunction with optical rotation, as in the study of Takagi and Isemura (1960) upon the alkaline denaturation of Takaamylase A. A recent example of the plural approach to protein structure is provided by the work of Edelhoch and Metzger (1961) upon thyroglobulin, in which they employ sedimentation, light scattering, viscosity, optical rotation, and polarization of fluorescence (Steiner and Edelhoch, 1961), as well as studying the kinetics of its denaturation (Metzger and Edelhoch, 1961). This protein, which can be dissociated into subunits prior to denaturation and is further fragmented by more drastic conditions, would appear to have a moderate helical content judging from the low A,, 225-235 mp, and [a1689 of - 63", values which decrease to 214 mp and - 99" in 9 M urea and increase to 275 mp and -39" in 2-chloroethanol. It is evident that monochromatic rotations are a useful adjunct to any study of protein structure, but a final illustration of the insight that rotatory dispersion can provide may be seen in the conformational analysis of chymotrypsinogen activation. Neurath et al. (1956) have found an exact correlation between the rate of activation of both chymotrypsinogen and trypsinogen as measured by the enzymatic activity of their products and the rate at which their specific rotations become more positive, a change which Neurath and Dixon (1957) suggest represents an increase in helical content. By dispersion measurements on chymotrypsinogen and r-chymotrypsin, Imahori et al. (1960) have demonstrated that a twofold increase in helical content, from 12 to 24%, indeed occurs. They are thus able to esti-
528
PnTER URNES AND PAUL DOTY
mate that the cleavage of a single peptide bond of the precursor, which has been shown t,o be the primary chemical event in the activation (Neurath and Dixon, l957), lcads to the formation of five or six turns of helix in the functioning enzyme.
I . Collagen The chemistry and physical properties of the collagen cIass of fibrous proteins form the subject of a comprehensive review in this volume by Harrington and von Hippel. Only a brief mention will accordingly be made of the salient points concerning the relation of its structure to optical rotation. The amino acid composition of collagen distinguishes it from all other proteins, for approximately every third residue is glycine and about one in four is either proline or hydroxyproline. The unique native structure of collagen, a triple-stranded helix proposed by Ramachandran and Kartha (1955) from X-ray diffraction data on the solid state, is explicable in large part by the properties of polypeptides containing these species of residue. The models of polyglycine (Crick and Rich, 1955) and poly-L-proline IT (Cowan and McGavin, 1955) based upon X-ray studies indicate that a chain with a specific sequence of these three residues can adopt a conformation characteristic of a single strand in collagen with hydrogen-bonding capabilities that make possible a stable unit of three strands (Rich and Crick, 1955). Each chain is arranged in a somewhat extended left-handed helix very similar to poly-~-proline11, and it fits into the collagen structure in a very gradual right-handed spiral groove provided by the other two chains. Hydrodynamic and light-scattering studies by Boedtker and Doty ( 1956) have shown that a variety of collagen, ichthyocol, from carp swim bladder behaves as a rigid rod in solution. If ichthyocol is heated to form gelatin, the product of collagen denaturation, the same methods indicate that three subunits are formed. These findings hence substantiate the three-stranded model for collagen in the solid state and furnish evidence that this same helical arrangement exists in solution. The optical rotatory dispersion of this unique type of helix is strikingly different from that of globular proteins and other fibrous proteins likc tropomyosin. As shown by Cohen (1955b), the dispersion is simple with a A, value of about 205 mlc and is characterized by a specific rotation, [ a ], ~ of about -350" at 11°C (Table XVI). Upon mild heating to 30"C, there is no alteration in A, , but the specific rotation rises to about - 1 lo", thus exhibiting a rhange in a direction opposite to that customarily found upon denaturation. While the rotatory properties of gelatin above 30°C are typical for a disordered chain, those of the native protein resemble the dispersion of poly-L-proline I1 (see Section 111, F ) , which has a large negative
OPTICAL ItOTATION AND 1'HOTlGIN CONPOHMATION
5fL9
[a]M0 of about -540' (Kurtz et al., 1956) that rises upon transformation to poly-L-prolineI and simple dispersion with a A, value of 206 mp that remains nearly constant over the first half of the transition. Upon cooling to 10°C, the specific rotation of gelatin drops toward the native value to about -300', again without change in A,. The balance of the evidence, as, for example, from the lack of concentration dependence of this reverse process, indicates that single gelatin chains assume a conformation similar to poly-Lproline 11. It therefore appears that the rotatory properties of collagen are largely determined by the left-handed helix characteristic of individual strands rather than hy the much more gradual right-handed helix that the three strands togcthcr describe. It is furthermore evident that rotatory dispersion in sensitive to different helical arrangements of a polypeptide chain, for the dispersions of the distinctive conformations of collagen and poly-L-proline I1 differ in almost all respects from that of the right-handed a-hclix that alniost surely exists in most proteins and polypeptides.
TABLE XVI Optical Iiotator?/ Properties of Collagen and Gelatin*
Collagen Gelatin
HzO, 0.15 M citrate buffer, pH 3.7, 11°C HzO, 0.15 M citrate buffer, pH 3.7, 40°C
-350 -110
205 205
* (Cohen, 1955b.)
J. Cotton hj$ects The measurement by Simmons et al. (1961) of a far ultraviolet Cottou cffect associated with helical polypeptides has already been cited in connection with the rotatory properties of the peptide bond (see Section 11, D ) . Another set of investigations has focused on the optical act.ivity which a symmetrical group, either a side chain, prosthetic group, or dye, assumes as a result of its interaction with an asymmetric protein or polypeptide. Blout and Stryer (1959) and Stryer and Blout (1861 ) have demonstrated that optically inactive dyes such as acridine orange, acriflavine, and pseudoisocyanine bound to helical poly-L-glutamic acid display striking Cotton effects a t one or more of their characteristic absorption bands, and that these disappear if the helix is disrupted, even though the dye remains att,ached. The signs of t.hese induced Cotton effects are, moreover, a function of helical sense, for they are reversed with poly-D-glutamic acid. Although these findings suggest an analogy for isolated side-chain chromophores in helical polypeptides, the asymmetric stacking of dye molecules attached to the polypeptide has not been ruled out.
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PETER URNES AND PAUL DOTY
I n the protein subunits of tobacco mosaic virus, Simmons and Blout (1960) report a small rotatory anomaly near 280 mp which they suggest may reflect oriented aromatic side chains. Ulmer et al. (1961) have found a similar deflection at 275-285 mp in the dispersion of alcohol dehydrogenase, as has Jirgensons (1961f) at 290-300 mp for glutamic acid dehydrogenase. The dispersions of these enzymes fail to display simple Drude behavior, a feature presumably attributable to these deflections. Although heme is not entirely a symmetrical structure, the small differences among the groups a t its periphery may not endow its principal transitions with optical activity. The finding of Cotton effects a t the characteristic heme absorption bands in proteins therefore suggests that this planar ring gains optical activity as a result of asymmetric binding. Eichhorn and Cairns (1958) have measured a Cotton effect near 560 mp in both oxidized and reduced cytochrome c. Cotton effects have been found a t the heme bands in catalase, myoglobin, hemoglobin, and sickle cell hemoglobin, as previously cited (Section IV, F). As is discussed by Ulmer and Vallee (1961), yeast alcohol dehydrogenase can be inhibited through chelation of its cofactor, zinc. Since the symmetric molecule 1,lO-phenanthroline becomes asymmetric upon coordination with zinc, there is reason to think that its inhibition of yeast alcohol dehydrogenase will be accompanied by the appearance of Cotton effects at its absorption bands. They find a negative Cotton effect a t 289 mp with smaller anomalies near 327 and 342 mp, all wavelengths associated with the enzyme-chelate complex. They also suggest that the environment of the zinc atom, which may participate in the stereospecific transfer of hydrogen to diphosphopyridine nucleotide, is asymmetric and hence can contribute to the Cotton effect. In a sequel to this study that has already been discussed in Section IV, F , Ulmer et al. (1961) have clearly shown that negative Cotton effects develop at the ultraviolet absorption bands of reduced diphosphopyridine nucleotide, the cofactor of this enzyme, and its analogues as they attach to the protein. They are also able to correlate this phenomenon, which they ascribe to asymmetric binding a t the active site, with the effects of competitive inhibition. Unfortunately, many of the observations of Cotton effects in proteiiis must, in the absence of spectrophotometric details, be viewed with some skepticism. This caution arises from the ease with which rotatory artifacts may be produced in regions of high absorbance. Winkler and Markus (1959) found that the unusual rotatory dispersions observed in azo-dye complexes of human serum albumin between 550 and 750 mp (Markus and Karush, 1958) were attributable to stray light polarized by reflection within the cell housing and which the opacity of dye solutions allowed to predominat>e.It has more recently bccn found (Urnes et al., 1961b) that stray light in the optical path can give rise to artifacts at absorption bands that simulate
OPTICAL ROTATION AND PROTEIN CONFORMATION
53 1
Cotton effects and hence can be systematically misleading. In the course of measurements to determine if Cotton effect,sappear at the tyrosyl absorption band as a copolymer of 5 % L-tyrosine with L-glutamic acid becomes helical (see Section 111, F ) , deflections observed near 285 mp at high absorbance and initially thought to signify an optically active chromophore were found to disappear at lower concentrations. Since any search for marginal Cotton effects will involve the use of high concentrations of material, the mechanism producing these spurious deflections has therefore been investigated. If the spectrum of this copolymer is reproduced by placing p-cresol, the side chain of tyrosine, in one cell in series with poly-L-glutamic acid in another, similar deflections appear at 276 mp as the absorbance of the p-cresol solutions is raised above 2, as illustrated in Fig. 15. The deflections at absorbances 4, 5 , and 6 can, in the absence of the poly-L-glutamic acid dispersion, be easily mistaken for Cotton effects associated with the chromophore. Since the chromophore, p-cresol, is itself optically inactive and is physically separate from the rotator, the deflections must be instrumental in origin. The explanation for this phenomenon is that solutions of high absorbance eliminate light for which the monochromator is set while permitting stray light outside the pass band to reach the analyzer and give rise to rotations characteristic for those wavelengths. If the slope of the true dispersion curve is sufficiently steep, as it is for poly-L-glutamic acid even in its disordered form, then these stray rotations will produce an apparent deflection at the center of the absorption band. Since these deflections are attenuated by an interference filter, the effect of which is indicated by the dashed line in Fig. 15, the characteristics of both the monochromator, in this instance a Beckman DU instrument attached to a Rudolph 200s polarimeter, and of the light source, here a General Electric AH-6 mercury arc with strong emission bands to the high wavelength side of the phenolic absorption band, are the factors which set a limit to the absorbances that can be safely employed. The limit with this instrument thus appears to lie between absorbances of 2 and 3 near 280 mp. As shown in Fig. 16, artifactual deflections are also observed at wavelengths at which the lamp fails to emit light, in this case the region of resonance absorption by the mercury itself between 255 and 260 mp, a situation identical with absorption by a chromophore in the optical path. A second deflection is obtained with poly-L-glutamic acid in the random coil as the peptide absorption band below 250 mp is entered. Since this deceptive effect, which will be more marked in the steeper dispersions characteristic of helices, can at lower wavelengths obscure the authentic Cottoneffect feature observed at 233 mp by Simmons el al. (1961) in helical polypeptides and proteins (Fig. 4), and since these authors propose the amplitude of this
532
PETER TTRNES A N D PAUL DOTY
feature as a measure of helical content (see Section IV, D),it is essential that the absorhances of solutions prepared for this type of conformational analysis he below the critical limit, which in this region of t,he spectrum may be less than an absorbance of 2. As a paramount criterion of the authenticity of Cotton effects in all re-2.0
-3.0
Cio
-4.c
-5.c
X (mp)
FIG.15. Rotatory artifacts t h at simulate Cotton effects a t a n absorption band. The dependence of the rotatory artifact on absorbance of p-cresol solutions placed in series with the same poly-L-glutamic acid solution is shown. The concentration of p cresol was adjusted to give the total absorbance of chromophore plus polypeptide background that appears with each curve. The rotator, poly-L-glutamic acid, was a t concentration of 0.5% a t p H 7.0 in a 10-cm cell. The rotations are those actually observed, (Y, in degrees. The rotatory dispersion a t An6 2' coincides almost exactly with th a t for the polypeptide alone, so that it has been omitted from the figure. At A276 4,an interference filter, f , with maximum transmission between 280 and 285 mp, was placed in the optical path. The absorption spectrum, in arbitrary units, is typical of p-cresol plus poly-L-glutamic acid background. The emission spectrum is represented in arbitrary units, uncorrected for detector response. (Urnes et al., 1961a.)
OPTICAL ROTATION A N D PROTEIN CONFORMATION
533
gions of the spectrum, it is therefore suggested that they be submitted to a Beer’s law type of test, which has its analogue for optical rotation in Biot’s law. If the effect is measured with two different amounts of material in the -2. ROTATORY
-4 ao
-6.1
-8.1 ABSORPTION SPECTRUM
EMISSION
SPECTRUM
240
260
280
300
A(mp)
FIG. 16. The appearance of rotatory artifacts both a t the minimum in the emission spectrum of the mercury arc and as increasing polypeptide absorption is encountered below 250 mp. The rotations are those actually observed, a, in degrees, for a 0.6% poly-L-glutamic acid solution at pH 7 in a 10-cm cell. The absorption spectrum of poly-L-glutamic acid and the emission spectrum of the arc, uncorrected for detector response, are in arbitrary units. (Urnes et al., 1961b.)
light path and, when plotted as the specific rotation, shows no changes in amplitude, shape, or wavelength at which the characteristic Cotton effects features appear, then the effect is genuine. In view of the variety of instrumental factors that contribute to artifactual deviations from this rule, the accurate measurement of rotatory dispersion at absorption bands entails a systematic consideration of the absorption spectrum of the sample, the emission spectrum of the light source, and the characteristics of the mono-
534
PETER URNES AND PAUL DOTY
chromator. Since optical rotation at the peptide absorption bands is now becoming the focus of experimental endeavor, it is especially important that instrumental limits be given explicit attention, for stray light effects increase progressively as the wavelength is lowered.
V. SUMMARY Of the various physical properties of proteins, their optical rotatory dispersion is perhaps one of the easiest to visualize, not only because it is simply the variation of rotatory power with wavelength but also for the reason that dispersion curves in the spectral range usually encompassed are smooth, featureless, and almost invariably become increasingly negative as the wavelength is decreased. This very likeness would hamper empirical comparisons were it not for a mathematical form, the simple Drude equation, into which protein dispersion data may be cast, a form that on quite general grounds describes the dispersion of all substances in regions distant from optically active absorption bands. Adherence to this equation, which specifies a dispersion as simple, yields three parameters, A, , A , and [m']~, the optical rotation at some reference wavelength, that permit one to distinguish in quantitative fashion the dispersion of one protein from another. Yet these characteristics alone supply no information on the mutual orientation of the peptide bonds, the principal chromophores of a polypeptide chain that both govern its dispersion and define its conformation. It is rather through the qualitatively different dispersion displayed by synthetic polypeptides known to be helical, a type that fails to obey the simple Drude equation and hence is designated as complex, that protein dispersions may be interpreted in t'erms of a specifically helical array of peptide bonds. An equation developed from the theoretical dispersion of helical structures, the Moffitt equation, is capable of characterizing this complex dispersion by four parameters, again [ m ' ]together ~ with the coefficients a. and bo and the dispersion constant XO The dispersion of d l substances becomes complex as optically active absorption bands are approached. However, among the diverse organic structures and physical arrangements occurring in polypeptide systems, it appears that only the standard helix gives a dispersion that is unquestionably complex in the transparent visible spectrum and furthermore possesses the explicit features of a bo near -630 and a XOof 212 mp. By virtue of the capacity of the Moffitt form with these constants to describe accurately mixtures of helices with disordered chains, differences among rotatory characteristics of many globular proteins can be given a structural explanation by the presence of varying amount,s of a standard polypeptide helix, a conformation that gives every indication of being a right-handed a-helix. Other conformations with periodic order, helices of opposite sense, and the extended or @-forms,may occur, as well
.
OPTICAL ROTATIOK A N D PROTEIN CONFORMATION
635
as completely random yet compact arrangements of the chain, but it is the existence of standard helical regions that is best established by rotatory dispersion. The manner in which optical rotatory dispersion contributes to an understanding of secondary structure in proteins is thus founded on their similarity to synthetic polypeptides. Since a protein differs fundamentally from a synthetic polypeptide in its unique sequence of amino acids, and since its particular function is ultimately attributable to this genetically determined order, optical rotatory dispersion might appear to offer little to the study of biochemical specificity. However, in the course of experimental transformations of one conformation into another for purposes of rotatory characterization, the role of primary structure as a determinant of the stability and even the occurrence of helical regions has emerged. A particular pattern of residues may therefore predispose a protein to a unique arrangement of secondary structure which, if disrupted, leads to a loss of specific biological activity. Even though rotatory dispersion yields information chiefly on the fraction of residues in general conformations common to both proteins and synthetic polypeptides, it does furnish some clues to the individuality of a protein by locating it approximately on a scale of helical content and by offering a technique for correlating changes in secondary structure with alterations in function. A continued comparison of the rotatory properties of proteins with those of synthetic polypeptides will, moreover, both sharpen this pattern of analysis and bring theory to bear on protein structure, especially as new rotatory phenomena that reflect the conformational state of the peptide bond are discovered in the far ultraviolet spectrum. Helical regions can account for much of the folded, compact nature of globular proteins, although other features of their structure, disulfide bridges and the susceptibility of certain side chains to hydrophobic forces, may also be required for the rigidity they exhibit in aqueous solution. We may close by asking if helical regions have more than structural significance for biochemically active proteins. They may serve simply as a framework for the appropriate orientation of essential side chains at a reactive site. But it is also conceivable that a helical segment can mediate chemical reactions at a distance by the property of electron transfer through its crystallike structure, a possibility that has received theoretical formulation (Evans and Gergely, 1949; Wirtz, 1950). Although evidence favoring this function of the helix is meager (Bucher, 1953 ; Lumry, 1959), it has lately received experimental support in the work of Kopple et at. (1961) which suggests that helical segments in poly-L-glutamic acid indeed promote oxidation-reduction reactions of inorganic complexes. The assessment of helical content in proteins by optical rotatory dispersion may therefore illuminate their chemistry by providing structural evidence germane to their specific reactivity.
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Author Index Numbers in parentheses are reference numbers and are included t o assist in locating the reference where the authors' names are not mentioned in the text#.Numbers in italics refer t o the page on which the reference is listed.
A Abderhalden E., 225, $14, 316 Abitz, W., 109, 132 Abraham, E. P., 274, 275, 814, 318 Acher, R., 187(174), 196, 223, 314 Acs, G., 149(29), 191 Ailhaud, G., 181(149), 194 Aiman, C. E., 242, 316 Akabori, S., 41, 134, 312, 316' Albertson, N. F., 239, 314 Albrecht, G., 7, 127 Aldridge, W. N., 297, 314 Alkjaersig, N., 152(55), 192 Allison, H., 226, 230, 231, 319 Almquist, H. J., 497, 636 Altar, W., 478, 637 Altman, K. I., 98, 133 Ambrose, E. J., 18, 43, 187, 128, 428, 478, 480, 484, 636, 638 Ames, W. M., 95, 96, 97, 98, 128 Anastasi, A., 514, 640 Anderegg, J. W., 356, 396 Anderer, F. A., 223, 285, 286, 287, 314 Anders, G., 36, 37, 132 Ando, T., 174(131), 194 Andreeva, N. S., 42,93, 94,116,130,134 Anesey, J., 96, 97, 100, 107, 187 Anfinsen, C. B., 141(6), 1181(151, 155), 185(163, 164), 187(178, 1791, 189(186, 187), 190(155, 191, 192), 191, 194 196, 293, 294, 296, 310, 314, 319, 514, 526, 636,649 Anson, M. L., 173, 194 Applequist, J., 430, 434, 435, 446, 468, 469,472, 473, 474, 477, 481, 636 Aqvist, S. E. G., 181(155), 190(155, 192), 194, 196
Arlinghaus, R. B., 224, 318
Arndt, U. W., 354, 396 Arndt, W. T., 430, 452, 636 Aroskar, J. P., 147(21), 191 Asadourian, A., 431, 470, 471, 477, 510, 511, 636 Ascherl, A., 251, 319 Astbury, W. T., 12, 15, 36, 43, 75, 128, 427, 636 Atkin, W. R., 75, 118 Auer, P. L., 333, 348, 363, 396, 398 Austin, A. T., 273, 814 Axelrod, A. E., 171(120, 121), 199, 194
B Backes, J. V., 254, 314 Badger, R. M., 18, 44, 1.28 Bailey, J. L., 160(81), 161(81), 192 Bailey, K., 492, 493, 636, 640 Baker, F., 329, 396 Balls, A. K., 305, 307, 321 Bamberger, E., 271, 314 Bamford, C. H., 12, 13,128,425,427,428, 448, 452, 636 Banfield, W. G., 128 Barakat, M. Z., 249, 251, 256, 282, $14, 319
Barbu, E., 359, 396 Barnafi, L., 223, 316 Barnard, E. A., 188(181, 182), 196 Barr, G., 375, 376, 396 Barry, V. C . , 314, 314 Barton, A. D., 265, 318 Baudras, A., 481, 641 Baumann, C., 244, 317 Bautz, E., 314, $14 Bear, R. S., 2, 3, 4, 5, 6, 38, 42, 43, 44, 45, 46, 47, 53, 58, 59, 60, 61, 63, 68, 75, 91, 92, 128, 189, 13.4, 136
545
546
AUTHOR INDEX
Becker, E., 36, 37, 138 Becker, R., 117, 188, 463, 636 Beecham, A. F., 10, 16, 188 Beek, J., 33, 128 Beeman, W. W. , 356, 396 Beer, M., 53, 56, 128 Behrens, 0. K., 223, 252, 316 Beiler, Th., 271, 381 Bell, E. A,, 236, 514 Bell, P. H., 223, 316 Bello, J., 98, 123, 124, 125, 126, 188, 189 (190), 196 Belner, R. J., 377, 378, 398 Ben Abdeljlil, A., 143(14), 145(14), 146(22), 147(22), 152(60), 191, 192 Bender, M. L., 231, 316 Bendet, I. J., 340, 358, 398 Benoit, H., 325, 397 Benson, E. E., 484, 636 Bensusan, H. B., 71, 72, 188 Bentley, J. P., 65,99, 138 Benronana, G. , 181(148), 194 Berends, F., 235, 316 Berger, A., 14, 15, 17, 18, 21, 22, 24, 25, 26, 27, 28, 29, 30, 106, 107, 128, 133, 137, 138, 271, 310, 311, 312, 316, 318, 458, 461, 529, 640, 643 Bergmann, M., 36, 188, 232, 516 Bernal, J. D., 9, 188, 361, 396 Bernfeld, P., 143(15), 148(23), 191 Bernier, I., 281, 51.5 Bernstein, P., 171(118), 195 Betheil, J. S., 98, 128 Bettelheim, F. R., 154(66), 198 Beveridge, J. M. R., 33, 188 Beychok, S., 454,466, 484, 503, 515, 636 Biancheria, A,, 357, 396 Bigelow, C. C . , 106, 128, 514, 636 Bigwood, E. J., 283, 319 Bijvoet, J. M., 16, 188, 453, 643 Bird, G. R., 429, 431, 636 Birkheimer, C. A., 280, 316 Biscoe, J., 59, 138 Biserte, G., 296, 316 Biswm, A. B., 9, 10, 132 Bjornholm, S., 359, 396 Blackburn, S., 230, 316 Blake, C. H., 4, 5, 13-4 Blatt, H., 209, 211, 818 Blickenstaff, R. T., 276, 316
Block, R. J., 280, 316 Bloom, S. M., 474, 476, 481, 636 Blout, E. R., 15, 17, 18, 24, 25, 30, 73, 116, 199, 130, 353, 397, 403, 404, 421, 422, 423, 429, 431, 434, 435, 436, 440, 441, 442, 443, 446, 449, 450, 451, 454, 457, 458, 459, 460, 461, 465, 466, 470, 471, 474, 476, 477, 478, 479, 481, 484, 485, 490, 492, 493, 498, 503, 504, 505, 507, 510, 511, 515, 519, 529, 530, 531, 636, 637,638,639,640,641, 642,643
Blumenfeld, 0. O., 517, 636 Blumenthal, H., 270, 316 Bly, R. S., 276, 316 Roardman, F., 435, 442, 464, 465, 639 Bodanszky, M., 223, 236, 280, 316, 316 Bodo, G., 237, 380 Boedtker, H., 63,65,67,68,69,70, 71, 78, 79, 81, 82, 96, 102, 108, 126, f28, 325, 346, 355, 361, 362, 396, 528, 636 Bolduan, 0. E. A,, 59, 60, 61, 128 Bolling, D., 280, 316 Bon, W. F., 109, 133 Booth, F., 61, 136, 351, 396 Born, M., 405, 407, 415, 636 Bott, M. J., 109, 110, 136, 478, 642 Bottle, R. T., 513, 515, 643 Bove, J. L., 256, 317 Bowes, J. H., 95, 98, 189 Boyer, P. D., 234, 316 Boyer, R. F., 387, 399 Bozicevich, J., 289, 316 Bradbury, E. M., 18, 53, 93, 94, 109, 116, 129, 441, 448, 450, 451, 455, 456, 457, 465, 472, 637, 638 Bradbury, J. H., 325, 346, 353, 354, 397, 404, 429, 430, 474, 637 Bragg, J. K., 112, 138, 473, 483, 64.4 Bragg, W. L., 63Y Brand, E., 463, 638 Branson, H. R., 46, 136, 353, 398, 415, 425, 432, 648 Braunitzer, G., 223, 287, 297, 316, 321 Bresler, S. E., 65, 129, 496, 501, 513, 514, 526, 637 Brink, N. G., 142(9), 191 Brinkman, H. C . , 346, 396 Brockmann, H., 225, 314 Broersma, S., 332, 362, 396 Bromer, W. W., 223, 252, 316
AUTHOR INDEX
Brown, A., 65, 96, 136, 329, 355, 598 Brown, D. D., 272, 315 Brown, D. M., 162(88), 193, 314, 315 Brown, G. L., 3, 61, 136 Brown, J. R., 173(129), 194 Brown, L., 12, 128, 425, 427, 451, 452, 456, 536, 537
Brunauer, S., 91, 129 Bruylants, A., 231, 316 Bryan, W. P., 431, 637 Buchert, A. R., 280, 305, 320 Buchner, E. H., 124, 129 Buckles, R. E., 251, 315 Bueche, A. M., 346, 396 Bueche, F., 370, 372, 396 Bucher, T., 198, 203, 218, 218, 535, 637 Bull, H. B., 351, 396 Bungenberg de Jong, H. G., 108, 132 Bunville, L. G., 521, 643 Burge, R. E., 18,53,61,67,68,73, 74,75, 76, 78, 79, 81, 82, 83, 92, 93, 94, 103, 104, 109, 116, i29, 136 Burge, R. T., 16, 25, 129 Burgers, J . M., 332, 362, 396 Burgstahler, A. W., 242, 315 Butler, A. M., 209, 211, 218 Buzeell, J. G., 325, 334, 340, 351, 352, 356, 358,359, 396, 400 C
Cabrera, G., 98, 133 Cairns, J. F., 515, 530, 638 Calvin, M., 233, 315, 474, 476, 496, 637 Cammaroti, M. S., 265, 317 Cant, E. M., 12, 128 Cantoni, G. L., 266, 316 Carpenter, D. C., 105, 124, 125, 129 Carpenter, D. K., 348, 398 Carroll, W. R . , 152(53), 175(53), 176(53), 192, 296, 314 Cars, N. 96, 133 Caspar, D., 361, 396 Cavallini, D., 280, 319 Cecil, R., 160(80), I92 Cerf, R., 364, 367, 370, 374, 396 Cesari, M., 430, 5 4 Challenger, F., 265, 315 Champagne, M., 355, 356, 357, 396 Chandrasekhar, S., 417, 637 Chang, F. C., 276, 315
547
Chappelle, E. W., 256, 316 Charles, M., 143(14), 145(14), 162(85), 165(85), 166(85), 167(85), 171(126), 172(126), 191, 193, 194 Charlwood, P. A., 359, 996, 399 Chauvet, J., 187(174), 195, 223, 3i4 Choate, W. L., 296, 314 Chouteau, J., 484, 537 Christensen, L. K., 496, 637 Chun, E. H. L., 69,100,101, 102, 129 Cicirelli, J. S., 96, 101, 138 Cipera, J. D., 236, 315 Claesson, S., 388, 396 Clark, G. L., 59, 129 Clarke, H. T., 232, 315 Cochran, W., 43, 44, i29, 425, 537 Coe, J. R . , Jr., 381, 399 Cohen, C., 5,7,44,45, 73, 74, 77, 108, 110, 111, 129, 181, 137, 422, 423, 434, 435, 467, 487, 490, 492, 493, 497, 503, 505, 507, 528, 529, 531, 537, 642 Cohen, E., 139(1), 141(1,4), 142(1), 143(4), 150(35, 36), 151, 152(47, 48), 152(4, 62), 162(84), 163(1), 164(1), 169(1), 173(47, 48), 191, 192, 193 Cohen, J., 96, 97, 100, 109, 127, 137 Cohen, J. A., 161(89), 193, 235, 305, 315, 318
Cohen, L. A., 223, 225,226, 252,256, 257, 258, 259, 260, 261, 262, 263, 264, 272, 291, 293, 515, 319 Cohn, E. J., 198, 200, 218, 337, 391, 396 Cole, R. D., 160(81), 161(81), 192 Coleman, J. E., 174(134, 136), 194 Condon, E. U., 405, 407, 478, 637 Connell, J. J., 87, 129 Constantin, M. J., 177(143, 144), 194 Conway, B . E., 351, 396 Cooke, J. P., 190(192), f 9 5 Coombes, J. D., 456, 458, 474, 637 Coombs, T. L., 174(132, 133, 134, 135), 194
CopiE, M., 370, 399 Corey, E. J . , 252, 315 Corey, R. B., 7, 9, 10, 12, 16, 44, 46, 54, 59,61, i27, 129,136, 138, 353, 398, 415, 425, 427,428,432, 478, 641, 542 Cotton, A., 420, 537 Courts, A., 95, 96, 97, 129 Cowan, P. M., 8, 16, 17, 19, 25, 43, 44, 45,
548
AUTHOR INDEX
48, 49,51, 53,60, 61, 92, 116,129, 230, 461, 528, 637 Craig, L. C., 251, 291, 292, 294, 316, ,917 Cragg, L. H., 371, 399 Craig, P. N., 239, 249, 276, 316 Creeth, H. J., 356, 396 Crick, F. H. C., 12, 13, 14, 16, 25, 43, 44, 46,48,49,50,52,53,54,129,130, 136, 361, 396, 425, 427, 447, 528, 637, 648 Cristol, S. J., 239, 316 Cullis, A. F., 483, 504, 642 Cunningham, R. S., 41, 67, 68, 69, 84, 85, 87, 88, 90, 137 Csok, R., 198, 203, 218, 218
147), 181(148, 149), 186(169), 191, 192, 193, 194, 196
de Vernejoul, P., 155(67), 156(67), 158 (67), 196 Dickerson, R. E., 402, 452, 462, 466, 483, 504, 506, 640 Dickman, S. R., 141(5), 147(21), 149, 150(5), 151(5, 40), 152, 181(154), 190(154), 191, 194 Dickson, T. W., 412, 64i DiMarzio, E. A., 112, 131, 473, 638 Dinh Van Hoang, 159(79), 198 Dintzis, H. M., 355, 396, 398 Dittmer, K., 239, 316 Dixon, G. H., 152(45, 52), 155(52, 68), D 158(77), 160(52), 161(52), 168(68), 175(52), 192, 223, 298, 305, 310, 316, Dalgliesh, C.E., 12, 15, 128 513, 515, 527, 528, 641, 646 Daniel, E., 430, 457, 458, 474, 637, 642 Dixon, J. S., 223, ,916 Darmon, S. E., 12, 15, 128 Dixon, M., 216, 618 das Gupta, S. R., 105, 137 Davie, E. W., 157(71), 192, 303, 304, 316 Djerassi, C., 420, 421, 509, 637 Dobry-Duclaux, A. (also Dobry, A.), Davies, D. R., 402,452,462,466,483,504, 351, 352, 396 640 Donohue, J., 8,9,11,16,130,429,452,637 Davis, D. S., 223, 516 Dorfman, A., 65, 67, 184 Davi8, H. F., 229, 318 Doring, W., 117, 128 Davis, P., 125, 130 Doty, P., 63, 65, 67, 68, 69, 70, 71, 74, 76, Dayhoff, M. O., 356, 357, 396 78, 79, 81, 82, 96, 100, 101, 102, 104, De, P. K., 488, 493,495, 508,520,521, 643 108, 126, 127, 129, 130, 132, 134, 138, Deasy, C., 95, 1.90 186(166), 196, 325, 346, 350, 353, 354, Debabov, V. G., 41, 136 355, 358, 359, 396, 397, 403, 404, 409, DeBellis, R., 41, 130 412, 414, 415, 417, 423, 424, 429, 430, Debye, P., 346, 396 431, 432, 433, 434, 435, 436, 437, 439, Deffner, G. G. J., 71, 72, 138 440, 442, 445, 446, 448, 449, 450, 453, de Garilhe, M. P., 280, 316 454, 456, 457, 458, 463, 465, 466, 467, De Jarnette, E., 189(190), 196 468, 469, 471, 472, 473, 474, 475, 476, Delavan, L. A., 226, 230, 231, 319 477, 478, 479, 480, 481, 484, 486, 487, de LosB, C., 431, 470, 471, 474, 476, 481, 488, 489, 490, 492, 494, 495, 498, 499, 484, 510, 511, 636, 637 500, 501, 503, 504, 508,509, 510, 511, De Marco, C., 280, 319 513, 515, 517, 519, 521, 524, 527, 528, Derksen, J. C., 108, 109, 130, 133 530, 532, 533, 636, 637, 638, 639, 640, Derrien, Y., 209, 210, 819 Desnuelle, P., 143(14, 16, 17), 145(14), 641, 64% 543, 644 147(16), 148(16), 152(43, 44, 60), Downie, A. R., 21,24,27,28,130,433,434, 435, 439, 441, 448, 449, 450, 451, 456, 154(43,44,64,65), 155(67), 156(65,67), 456, 457, 458, 461, 465, 472, 474, 637, 157(64, 72), 158037, 75), 159(79), 638 161(65), 162(85, 86), 163(105), 164 (105), 165(85), 166(85), 167(72, 85), Drey, C. N. C., 280, 316 168(107, lOS), 170(116), 171(126), Dreyer, W. J., 157(73), 172(125), 192, 194, 310, 311, 318, 497, 627, 641 172(126), 176(142), 177(16, 17, 143, 144), 178(142, 145, 146, 147), 180(146, Drisko, R. W., 305, 319
AUTHOR INDEX
Drude, P., 406, 638 Dryden, H. L., 276, 316 Dumsha, B., 4, 5, 33, 35, 131 Dus, K., 298, 316 du Vigneaud, V., 223, 254, 255, 316, 316, 318, 319
E Eastoe, J. E., 31, 32, 76, 130 Ebata, M., 312, 316 Edelhoch, H., 446, 489, 527, 638, 641, 643 Edlbacher, E., 271, 317 Edmundson, A. B., 483, 638 Edsall, J. T., 6, 130, 198, 200, 218, 335, 337, 356, 358, 360, 361, 363, 391, 396, 397, 398, 399, 426, 429, 461, 402, 482, 483, 638, 641 Ehrlich, G., 350, 396 Eichhorn, G., 515, 530, 638 Eidinger, D., 33, 131 Einstein, A., 331, 336, 397 Eisenberg, H., 373, 397 Eldridge, J. E., 103, 108, 110, 123, 130 Ellenbogen, E., 236, 316 Elliott, A., 12, 13, 18, 43, 127, 188, 130, 416, 425, 427, 428, 429, 431, 433, 434, 435, 439, 441, 448, 449, 450, 451, 452, 455, 456, 457, 458, 465, 472, 474, 478, 479, 480, 481, 484, 636, 637, 638 Elliott, D. F., 226, 316 Elrod, H., 379, 398 El-Sadr, M. M., 249, 251, 282, 31.4 El-Wahab, M. F. A., 249, 251, 282, $14 Emmett, P. H., 91, 129 Endo, R., 388, 399 Endres, H., 33, 38, 98, 131 Engel, J., 103, 131 Engelbertz, P., 270, 320 Engle, R. R., 237, 305, 319 Ens, A., 359, 396 Erlanger, B., 463, 638 Esipova, N. G., 76, 78, 93, 116, 130 Eugster, C. H., 242, 316 Evans, M. G., 535, 638 Ewald, A. Z., 75, 130 Ewart, R. H., 379, 398 Eyring, H., 405,478,482,497,637, 638,640
F Fabre, C., 157(72), 167(72), 192 Falconer, J. S., 209, 211, 212, 218
549
Fankuchen, I., 361, 396 Fanselow, J. R., 110, 133 Fasman, G. D., 15, 17, 18, 24, 25, 26, 27, 30, 116, 129, 130, 133, 226, 316, 434, 435, 436, 441, 443, 451, 452, 455, 457, 458, 459, 460, 461, 474, 476, 478, 479, 481, 519, 636, 638, 640, 641 FaurB-Fremiet, M. E., 64, 130 Ferry, J. D., 103, 108, 109, 110, 113, 122, 123, 124, 125, 126, 130, 131 Fessler, J. H., 65, 99, 130, 132 Finogenov, P. A., 65, 129 Fischer, E., 225, 228, 270, 316 Fisher, E. H., 143(15), 148(23), 191 Fishman, L., 98, 133 Fitts, D. D., 405, 407, 415, 416, 451, 461, 465, 478, 509, 638, 639, 641 Fixman, M., 349, 399 Fletcher, A. P., 152(55), 192 Flodin, P., 294, 319 Flory, P. J., 75, 79, 80, 81, 97, 108, 111, 113, 114, 117, 118, 126, 130, 131, 325, 330, 347, 371, 397 Floyd, N. F., 265, 317 Folk, J. E., 152(49, 50, 51, 53), 174(137), 175(49, 50, 51, 53), 176(53, 140), 192, 194, 312, 380 Folts, C. M., 226, 267, 268, 269, 294, 318 Foss, J. G., 496, 526, 638 Foster, J. F., 359, 397, 400, 482, 486, 497, 505, 508, 522, 524, 641, 643, 644 Fox, T. G., Jr., 325, 347, 397 Fraenkel, G., 227, 316 Fraenkel-Conrat, H., 223, 280, 285, 287, 316, 318, 320 Francis, J. E., 235, 243,274, 275, 316, 319 Franklin, R. E., 361, 397 Fraser, R. D. B., 44, 45, 61, 62, 94, 130, 136, 136 Freese, E., 314, 314 Frenkel, S., 495, 513, 658 Frenkel, S. Y., 65, 129,496, 501, 513, 514, 526,637 Frensdorff, H. K., 359, 397, 497, 638 Fresco, J. R., 472, 638 Freter, K., 272, 316 Freudenberg, K., 415, 420, 640 Fridrichsons, J., 10, 16, 128 Fried, J., 280, 316, 316 Fried, M., 314, 316
550
AUTHOR INDEX
Goldstein, L., 431, 434, 435, 474, 477, 689 Goldstein, M., 369, 397 Golub, M. A., 497, 639 Golubow, J., 171(120,121), 193,19.4 Gonell, H. W., 75, 132 Goodman, L., 238, 316 Goodman, M., 416,435,442, 464,465,639 Goodwin, S., 256, 316 Goodwin, T. W., 280, 316 Gordon, A. H., 31, 131 Gordon, R. S., 123, 131 G Gorguraki, V., 489, 490, 516, 518, 640 Gabeloteau, C., 155(67), 156(67), 158 Gouinlock, E. V., 97, 131 Grabar, P., 125, 131 (67), 168(107, 108), 196, 193 Grafe, K., 232, 316 Gadd, J. O., Jr., 363, 399 Grannis, G., 230, 319 Gallagher, K. J., 116, 130 Gallop, P. M., 41, 64, 65, 67, 68, 69, 71, Grant, N . H., 142(12), 191 84,85,86, 87, 88,90,98, 101,118, 128, Grassmann, W., 33, 37, 38, 40, 41, 63, 75, 86, 98, 103, 131, 133 130, 131, 134, 13Y Gratzer, W. B., 423, 424, 432, 433, 435, Gans, It., 337, 397 457, 474, 477, 524, 527, 530, 533, 639, Gardner, C. S., 348, 396 Garrault, A., 64, 130 643 Green, A. A., 198, 199, 201, 202, 218 Garrett, E. R., 231, 316 Garrett, R. R., 75, 79, 80, 108, 113, 126, Green, F. C., 36, 37, 45, 136, 13Y Green, N. M., 172(124), 194 130,131 Greene, L. J., 150(37), 191 Gaskins, F. H., 381, 399,430, 646 Greenberg, D. M., 497, 636 Gatovskaia, T. V., 93, 116, 130 Greengard, H., 152(58, 59), 198 Gebhardt, E., 38, 133 Greenshields, R. N., 173(129), 194 Gehring-Muller, R., 223, 297, 316 Geiduschek, E. P., 403, 439, 463, 473, Greenstein, J. P., 224, 225, 228, 235, 236, 316, 31Y, 381 63Y, 642 Gregory, J. D., 292, 294, 317 Gergely, J., 535,638 Grisolia, S., 488, 6-40 Gerngross, O., 109, 126, 132 Geschwind, I. I., 106, 128, 223, 316, 514, Gross, E., 223, 226, 246, 247, 248, 250, 251, 252, 261, 267, 268, 269, 280, 281, 636 Gibbs, J. H., 112, 131, 473, 638 282, 291, 292, 293, 294, 295, 296, 314, Gish, D. T., 223, 281, 287, 316, 320 3ir, $18 Gladner, J. A., 152(50, 51, 53), 174(137), Gross, J., 4, 5, 31, 32, 33, 34, 35, 38, 58, 175(50, 51, 53), 176(53, 140), 193, 194, 59, 60,61, G2,63,64,65,71,76,78,99, 312, 320 103, 104, 119, 131, 132, 136, 136 Glazener, M. R., 265, 318 Grossman, M. I., 152(58, 59), 192 Glazer, A. N., 223, 318, 423, 638 Guidoni, A , , 155(67), 156(67), 158(67), Glazer, N., 4, 33, 35, 131 162(85), 165(85), 166(85), 167(85), Glegg, E., 33, 131 192, 193 Glick, R. E., 256, 319 Guidotti, G., 297, 317 Go, Y., 11, 134 Gundlach, H. G., 188(180), 196, 233, 26G, Godfrey, T. B., 381, 399 267, 268, 31Y Goering, H. L., 239, 316 Gustavson, K. H., 3,33,76,77,82,91,98, Goldberg, P., 377, 39Y 131, 132, 137 Goldschmidt, S., 257, 316
Friedberg, F., 279, 280, 316, 319 Friedman, E., 440, 477, 638 Fries, G., 33, 132 Frohwirt, N., 298, 318 Fromageot, C., 280, 316 Fu, S. C. J., 235, 316 Fujioka, H., 174(131), 194 Fujita, Y., 224, 316 Fuller, W., 55, 130 Fuoss, R. M., 350, 377, 397
AUTHOR INDEX
Guy, O., 162(85, 86), 163(105), 164(105, I%), 165(85, 106), 166(85), 167(85), 193
H Haber, E., 189(186), 190(191), 195 Haefele, L. F., 252, 316 Hafter, R., 33, 231 Hall, C. E., 38, 43, 46, 58, 61, 63, 67, 68, 127, 132, 136, 360, 361, 397, 431, 639 Halpern, A., 424, 432, 466, 643 Haltner, A. J., 332, 346, 355, 362, 397, 398 Ham, J. S., 415, 423,639 Hanby, W. E., 12, 13, 15, 128, 132, 416, 425, 427, 428, 429, 431, 433, 434, 435, 439, 441, 448, 449, 450, 451, 452, 455, 456, 457, 458, 465, 472, 474, 479, 481, 636, 637, 638 Hannig, K., 38, 40, 103, 131 Happey, F., 425, 427, 428, 636 Harington, C. R., 236, 317 Harker, D., 189(190), 196 Harkness, R. D., 65,99, 132 Harrington, W. F., 20, 21, 22, 24, 25, 26, 27, 28, 29, 73, 74, 77, 81, 82, 83, 86, 89, 90, 91, 104, 105, 106, 107, 108, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 132, 133, 134, 137, 186(165), 196, 251, 317, 356, 357, 359, 397, 461, 462, 477, 489, 513, 514, 526, 639, 642, 643 Harris, J. I., 171(117), 193, 223, 317 Harshman, S., 237, 305, 319 Hart, R. G . , 402, 452, 462, 466, 483, 504, 640
Hartley, B. S., 158(76), 161(90, 91, 92), 163(92,93), 169(76), 182,193,229,310, 318
Hashizume, H., 487, 527, 639 Hastewell, L. J., 123, 132 Haurowitz, F., 513, 515, 643 Hawn, C. V. Z., 360, 399 Hayward, B. J., 265, 316 Heidemann, E., 96, 133 Heidenhain, R., 148, 191 Heine, H. W., 256, 317 Heinke, B., 235, 321 Heller, W., 405, 407, 409, 412, 421, 509, 639 Hermann, K., 109, 126, 132
551
Hermans, J., 116, 132 Hermans, J. J., 350, 351, 373, 376, 378, 382, 397, 400 Hermans, J., Jr., 378, 397, 474, 476, 496, 504, 637, 639, 642 Herriott, R. M., 151, 152(42), 154(42), 191, 280, 318 Herskovits, T. T., 282, 287, 317 Herzog, R. O . , 75, 132, 337, 397 Hess, G. P., 169(111), 195, 307, 321 Hewitt, L. F., 502, 639 Heymann, E., 108, 132 Heyns, K., 36, 37, 41, 104, 121, 132 Highberger, J. H., 60, 61, 62, 63, 65, 71, 72, 99, 131, 132, 136 Hill, R. J., 297, 317 Hill, R. L., 223, 317 Hill, T. L., 473, 639 Hilschmann, N., 223, 297, 316 Hilse, K., 223, 297, 316 Hinman, R. L., 244, 317 Hirai, N., 109, 123, 132 Him, C. H. W., 141, 149(8), 181(153), 182(158, 159, 160), 191, 194, 196, 223, 294, 317,483,638 Hirsch, G. C., 153(61), 192 Hirschmmn, D. J., 280, 318 Hobom, G., 223, 297, 316 Hoch, F. L., 174(135), 194 Hormann, H., 33, 41, 98, 131, 132 H@yrup,M., 202, 219 Hodge, A. J., 63, 70, 71, 72, 132 Hoffman, J. D., 117, 1% Hofmann, K., 223, 226, 231, 235, 317 Hofmann, T., 168(109, 110), 193 Hofmann, U., 33, 38, 63, 131, 133 Hofstee, B. H. J., 171(119), 193 Hokin, L. E., 147(20), 150(34), 191 Holey&ovsk$, V., 163(94, 97, 99, 101), 193, 235, 320 Holleman, J. W., 296, 316 Holleman, L. W. J., 108, 132 Holleman-Dehove, J., 296, 316 Hollies, N. R. S., 355, 398 Holtzer, A. M., (also Holtzer, A.), 325, 346, 353, 354, 397, 404, 429, 430, 637 Holtzer, R. L., 151(39), 191 Holzwarth, G. M., 423, 424, 639 Honnen, L., 36, 37, 45, 136, 137 Horn, P., 495, 513, 638
552
AUTHOR INDEX
Houwink, R.,329,397 Howard, J., 273, 314 Howes, E.L.,41, 130, 134 Hoyt, B. L.,71, 72, 128 Hudson, H., 410, 641 Hufford, D.L.,243, 3f7 Huggins, M.L.,43,54,132,328,597,452,
514, 515, 516, 517, 518, 522, 530, 659,
640 Jonsson, B.,190(192), 106 Johnson, C.,427,640 Johnson, P.,356, 357, 359, 397 Johnson, R.C.,251,3f6 Johnston, J. P., 101,133 JollBs, P.,281, 316 639 Jones, R . A. Y., 227, 317 Hughes, E. W., 9,10, 132 5008, G.,409, 640 Hughes, W.L.,198, 218 Jopling, D.W.,123, 133 Hughes, W.L., Jr., 356, 397 Jorpes, J. E., 140(2), 191 Hunt, H. D.,415, 639 Joyce, B.K., 488, 640 Hurley, R.,31, 135 Hynes, R. D., 67, 68, 73, 74, 75, 76, 78, Junqueira, L. C. U., 153(61), 192
79,81, 82,83, 103,104, 129 I
Idelson,
K Kaesberg, P., 59, 133, 356,396
M.,429, 434, 446, 465, 470, 471, Kallio, R.E.,41, 137
474,476, 498,504,636,638,639 Ikeda, Y.,370,397 Ikenaka, T.,486, 489, 490, 513, 514, 516, 518, 640 Illig, R.,337, 397 Imahori, K.,424,432,435, 436, 446, 453,
Kalnitsky, G.,186(167), 196 Kappeler, H.,223, 519 Karlson, R. H.,434, 436, 441, 450, 451, 457, 474, 656,640 Kartha, G.,47,51,54,56,136, 528,648 Karush, F., 359, 398, 513, 515, 526, 530, 641 Kassel, B., 162(87), 163(87, 102, 103,
454, 466, 471, 474, 477, 479, 480, 484, 486, 487, 488, 489, 492, 494, 495, 498, 637,639, 643 104),198 499,509,510,527,532, Katchalski, E.,14, 15, 17, 18, 20, 21, 22, Inagami, T., 171(122), 194 24, 25, 26, 27, 28, 29, 106, 107, 128, Ingalls, E.N.,325, 400 133, 137, 271, 518, 403, 430, 431, Inouye, J. M.,232, 316 435, 456, 457, 458, 459,460,461, 473, Irreverre, P.,224, 300, 517, 320 474, 477, 529, 637, 639, 640, 641, 645 Iselin, B.,223, 319 Katchalsky, A., 350, 398 Isemura, T., 527, 643 Katritrky, A. R . , 227, 317 Ishii, S.,291, 292, 517 Katsoulis, M.,270, 319 180, K . , 465,473,474,475,47G,504,644 Katsoyannis, P.G . , 222, 223,316,317 Ivy, A. C., 152(58, 59), 198 Katz, J. R., 105, 108, 109, 110, 124, 125, Iwai, K.,226, 237, 917 135 Izumiya, N.,240, 241, 317 Katz, S.,358,396 J Kaufman, D.L.,305, 316 Kauzman, W.,189(188), 196, 35'3, 397, Jackson, D.S.,65,99, 139 398, 403, 405, 416, 423, 438, 482, 496, Jackson, S. F.,3,45,61,62,133, 136, 136 497, 521, 524, 638,640,643 Jacobsen, C. F.,154, 192 Kawanishi, Y., 174(131), 19.6 Jaenicke, R.,486, 514, 515, 64.9 Jakus, M.A., 38, 43, 46, 58, 61, 139, 136 Kay, C.M., 358, 398, 487, 491, 492, 493, 513, 640 James, S., 280, 305, 320 Kay, L. M., 37, 45, 137 Jansz, H. S.,235, 916 Kazakova, 0.V . , 41, 153 Jeffery, G.B., 332, 363, 397 Kazenko, A., 152(46), 154(46), 163(46), Jenden, D.J., 209, 211,212, 918 199 Jirgeneons, B.,403, 485, 486, 487, 488, 489, 490, 491, 497, 502, 503, 508, 513, Keech, M.K., 72, f38
AUTHOR INDEX
Kegeles, G., 357, 396 Keil, B., 163(94, 95, 96, 97, 98, 100, 101), 1 g3, 280, 317 Keith, C. K., 152(46), 154(46), 163(46), 192
Keller, P. J., 139(1), 141(4), 142(1), 143(4), 150(35, 36), 151, 152(47, 48), 153(4, 62), 163(1), 164(1), 169(1), 173(47,48, 128), 191, 192, 194 Kelly, F. C., 3, 61, 136 Kenchington, A. W., 95, 126, 133 Kendrew, J. C., 3, 5, 6, 43, 133, 361, 396, 402, 452, 462, 466, 483, 504, 506, 640 Kenner, G. W., 280, 316 Kenton, R. H., 95, 98, 129 Kezdy, F., 231, 316 Kies, M. W., 272, 316 Kimball, G. E., 405, 638 Kimmel, H., 230, 319 Kirk, D., 71, 131 Kirkwood, J. G., 198, 219, 333, 346, 348, 356, 363, 370, 398, 399, 415, 416, 452, 461, 465, 478, 668, 640, 641 Kitai, R., 184(162), 196, 223, 319 Kliimbt, H.-D., 244, 317 Klein, L., 41, 137 Klemperer, E., 429, 435, 437, 446, 471, 474, 476, 477, 480, 494, 495, 498, 499, 515, 637, 639, 640 Kline, E. S., 31, 133 Klug, A., 361, 397 Klyne, W., 421, 509, 637, 640 Knight, C. A., 223, 289, 31Y, 320 Knilling, W. von, 251, 319 Kny, H., 272, 317 Kolb, J. J., 265, 268, 320 Kolthoff, I. M., 514, 640 Konigsberg, N., 256, 317 Konigsberg, W., 297, 317 Konishi, E., 477, 478, 479, 519, 643 Konno, K., 98, 133 Kopple, K. D., 535, 640 Koshland, D. E., 270, 319 Kossel, A., 271, 317 Kostka, V., 163(98, 101), 193 Koszalka, T. R., 243, 317 Kounina, 0. V., 70, 91, 103, 136 Kozarenko, T. D., 41, 136 Kraemer, E. O., 110, 133, 327, 328, 398 Kramer, H., 5, 133 Krasny-Ergen, W., 351, 398
553
Kratky, O., 58, 133 Krieger, I. M., 376, 379, 398 Krigbaum, W. R., 348, 398 Kroepelin, H., 377, 398 Kroner, T. D., 36, 37, 45, 133 Kropf, R. B., 147(21), 191 Kucera, J. J., 105, 125, 129 Kudar, H., 337, 397 Kuntzel, A., 79, 96, 133 Kunzle, O., 350, 398 Kuhn, H., 332, 363, 370, 398 Kuhn, K., 33, 38, 63, 131, 133 Kuhn, W., 332, 350, 363, 370, 398, 407, 410, 415, 420, 604 Kulonen, E., 65, 67, 134 Kunitz, M., 151, 152(42), 154(42), 191, 280, 318 Kunst, P., 161(89), 193, 305, 318 Kurata, M., 348, 398, 400 Kurtz, J., 14, 15, 17,18,26,27,28,29, 128, 133, 312, 316,458,461, 529, 640 Kushner, V. P., 496, 801, 513, 514, 526, 637
Lakshmanan, B. R., 55, 56, 133 Lan, H. D., 280, 316 Lande, S., 223, 317 Lang, J., 244, 3i7 Langheld, K., 257, 317 Lansing, W. D., 327, 398 Laskowski, M., 151(41), 152(46), 154 (46), 162(87, 88), 163(87, 102, 103), 181(150),191, 192, 193, 194 Laskowski, M., Jr., 151(41), lQf, 282, 287, 317, 325, 346, 355, 359, 399 Latham, H. G., Jr., 270, 319 Lauffer, M. A., 340, 358, 398 Lauritzen, J. I., 117, 133 Lavine, T. F., 265, 317 Lawler, H. C . , 223, 316 Lawson, W. B., 171(123), 194, 223, 226, 244, 245, 246, 247, 248, 250, 251, 252, 260, 261, 267, 268, 269, 276, 280, 281, 282, 291, 294, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 317, 318, 320
Leach, A. D., 31, 32, 130, 133 Leach, 8. J., 229, 318, 403, 640 Leblond, C. P., 33, 131 Lee, G. R., 230, 316
554
AUTHOR INDEX
Lee, R. H., 145(18), 191 LeGette, J., 37, 45, 137 Legler, G., 41, 104, 121, 132 Lenhoff, H. M., 31, 133 Lenormant, H., 481, 484, 636, 637, 540, 641 Leonard, W. J., Jr., 486, 505, 508, 522, 524, 641 LBonis, J., 497, 517, 636, 641 Leplat, G., 64, 133 Lerner, A. B., 223, 317 Leung, Y. C., 8, 9, 11, 16, 133 Levy, M., 98, 133, 134, 225, 318, 320 Lewis, J. C., 280, 318 Lewis, M. S., 74, 76, 100, 101, 102, 103, 104, 134, 136, 265, 318 Lewis, U. J., 142(9, lo), 191 Li, C. H., 223, 316 Li, T.-K., 516, 530, 643 Liebold, B., 223, 297, 316 Liener, I. E., 169(112, 113, 115), 193 Lifson, S., 473, 641 Light, A., 223, 318 Likins, R. C., 4, 136 LinderstrZm-Lang, K. U., 86, 134, 222, 318, 484, 499, 510, 636, 641 Lindley, H., 6, 134, 312, 318 Lindley, N., 519, 641 Lipman, F., 149(29), 191 Lippoldt, R . E., 446, 638 Listowsky, I., 435, 442,464, 465,639 Little, K., 5, 133 Liu, T., 223, 31'7 Loder, B., 275, 318 Loeb, G. I., 325, 340, 355, 356, 357, 398, 504, 648 Logan, M. A., 41, 137, 224, 318 Lorimer, J. W., 67, 68,138 Love, P., 256, 317 Lovelace, F. E., 105, 125, 129 Low, B. R., 426, 429, 461, 641 Low, B. W., 356, 398 Lowenstein, A,, 21, 188 Lowey, S., 325, 346, 397 Lowry, T. M., 409,410, 412, 421, 641 Lucas, C. C., 33, 188 Luck, J. M., 256, 516, 317 Lukin, M., 98, 131 Lumry, R., 535, 641 Lundberg, R. D., 416, 448, 449, 450, 465, 637, 641
Lurrati, V., 356, 398, 430, 641 Lynn, L. T., 285, 518
Mc McDonald, T. R. R., 451, 456, 637 M'Ewen, M. B., 65, 67, 134 McGarr, J. J., 36, 37, 45, 133 McGavin, S., 8, 17, 19, 43, 45, 49, 51, 53, 92, 116, 189, 130, 461, 528, 637 MacInnes, D. A., 356, 357, 396 McIntyre, A. D., 117, 130 MacLennan, J. D., 41, 130, 134 McMeekin T. L., 517,641 McPhee, J. R., 160(80), 192 MacRae, T. P., 94, 130 McRorie, R. A,, 265, 318
M Macheboeuf, M., 359, 396 Maeda, A., 486, 641 Malcolm, B. R., 12, 188, 230, 416, 427, 429, 433, 434, 435, 439, 448, 449, 450, 451, 452, 455, 479, 481, 638 Mandelkern, L., 75, 79, 134, 337, 339, 340, 343, 358, 362, 391, 392, 399 Mandl, I., 41, 130, 134 Marchis-Mouren, G., 143(13, 14, 16, 17), 144(13), 145(13, 14), 147(16), 148 (l6), 177(16, 17, 143, 144), 178 (145), 191, 194 Margoliash, E., 298, 318 Mark, H., 329, 398 Marko, A. M., 65, 99, 138 Marks, M . H., 4, 5, 134 Markus, G., 513, 515, 526, 530, 641, 643 Maron, S.H., 376, 377, 378, 379, 398 Maroux, S., 162(86), 170(116), 171(126), 172(126), 177(116), 193, 194 Marsh, M. M., 487, 513, 640 Marsh, R. E., 7, 8, 9, 11, 16, 133, 134, 428, 641 Marshall, J. M., 150(38), 191 Martin, A. F., 328, 398, 399 Martin, A. J. P., 181(152), 194 Martin, A. V. W., 45, 61, 62, 136 Martin, C., 171(120, 121), 193 Martin, G. R., 72, 103, 134, f36 Martin, K., 257, 316 Massey, V., 310, 318 Masson, F., 430, 641 Mather, A. N., 266, 380
AUTHOR INDEX
Mathews, M. B., 65, 67, 134 Mathieson, A. McL., 7, 8, 10, 11, 16, 228,
555
268, 269, 283, 293, 294, 295, 300, 312, 31Y) 319,320
Moore, W. J., 9, 10, 132 Morel, J., 125, 131 Matoltsy, A. G., 5, 131 Morgan, R. S., 60, 61, 128 May, W., 270, 320 Maybury, R. H., 152(52), 155(52, 68), Morrill, G. A., 151(40), 181(154), 190 (154), 191, 194 160(52), 161(52), 168(68), 175(52), Morris, A. J., 141(5), 149, 150(5), 151(5), 192, 513, 515, 642 152, 191 Mazourov, V. I., 65, 70, 91, 103, 134, 136 Morton, R. A., 280, 316 Mecham, D. K., 234, 318 Moscowitz, A., 407, 419, 420, 641 Mechanic, G., 98, 134 Mosimann, H., 97, 134 Meedom, B., 156, 158, 192 Moss, J. A,, 95, 129 Meggy, A. B., 14, 134 Moubasher, R., 256, 319 Mehl, J. W., 332, 341, 390, 398 Mudd, S. H., 266, 318 Meiboom, S., 21, 128 Mueller, J. M., 254, 318 Meienhofer, J., 223, 316 Meilman, E., 41, 65, 67, 71, 86, 98, 118, Muller, R., 223, 297, 516 Muir, H. M., 65, 99, 132 150, 131, 134, 137 Muirhead, H., 483, 504, 642 Melnick, S . C., 33, 134 Meloun, B., 163(94, 96, 97, 98, 100, 101), Muller, T. C., 535, 640 Murakami, H., 416,478,502,541 193 Murakami, M., 291, 318 Mergenhagen, S. E., 72, 134 Murayama, M., 515, 641 Merigan, T., 310, 311, 318 Murjahn, R., 270, 320 Mernan, J. P., 360, 361, 399 M u t t , V., 140(2, 3), 191 Metzger, H., 489, 527, 638,641 Meyer, K. H., 11, 134, 254, 318 N Meylink, B., 124, 129 Michaelis, R., 270, 320 Nace, H. R., 276, 318 Michaels, S., 41, 134 Nagai, Y., 41, 64, 154 Michl, H., 223, 320 Nagel, F., 257, 316 Mihalyi, E., 86, 87, 132, I S 4 Nageotte, J., 61, 134 Mikes, O., 163(94, 99, 101), 195, 235, 320 Nakajima, A., 504, 642 Miller, K. D., 152(54), 192 Narahashi, Y., 291, 318 Miller, R. R., 535, 640 Narita, K., 226, 318 Millionova, M. I., 42, 94, 134 Naughton, M. A., 229, 318 Mitchell, J. C., 434,465, 478, 641 Neet, K. E., 285, 318 Mitchell, R. F., 380, 398 Neiman, R. E., 108, 134 Miyazawa, T., 478, 641 Neuberger, A., 9, 65, 99, 132, 134 Modderman, R. S . T., 108, 132 Neuman, R. E., 31, 32, 33, 134 Moffitt, W., 410, 412, 413, 414, 415, 41G, Neurath, H., 139(1), 141(1, 4, 7), 142(1), 418, 420, 423, 432, 434, 437, 452, 478, 143(4), 152(7, 45, 47, 48, 52), 153(4, 62), 154(7), 155(52, 68), 157(71, 73, 493, 641 74), 158(77), 160(52), 161(52), 163(1), Moggridge, R. C. G., 236, 317 164(1), 167(74), 168(68), 169j1, 74), Montroll, E. W., 96, lS4 172(124, 125), 173(7, 47, 48, 128, 129), Mooney, M., 379, 398 174(132, 133), 175(52), 191, 192, 194 Moore, B. W., 145(18), 191 298, 303, 304, 305, 310, 316, 358, 398, Moore, S., 149(33), 161(83), 162(83), 181 497, 513, 515, 527, 528, 541, 642 (153), 182(157, 158, 160), 183(157), 185(157), 188(180, 183), 189(157), 191, Newman, M. S., 254, 518 193, 194, 195, 223, 233, 265, 266, 267, Newton, G. G. F., 274, 275, 314,318 134
556
AUTHOB INDEX
Passaglia, E., 325, 371, 598 Pasteur, L., 407, 641 Patchett, A. H., 275, 277, 318 Patchornik, A., 223, 226, 230, 231, 232, 234, 245, 246, 247, 248, 250, 251, 252, 260, 261, 271, 272, 280, 281, 282, 291, 298, 303, 316, 318, 319, 381 Paulikhina, L. V,, 65, 156 Pauling, L., 6, 9, 10, 12, 16, 19, 44, 46, 53, 54, 129, 136, 353, 398, 415, 425, 427, 428,432,478, 641,648 Pechkre, J.-F., 152(45, 52), 155(52, 68), 157(74), 160(52), 161(52), 167(74), 168(68), 169(74), 175(52), 192, 298, 542 316, 513, 515, 642 Northrop, J. H., 151, 152(42), 154, 191, I’edersen, K. O., 336, 337, 356, 391, 599, 280, 318 497, 642 Nozaki, Y., 521, 643 Peerdeman, A. F., 16, 128 0 Peller, L., 473, 64.9 Peng, Chia-Mu, 65, 67, 136 O’Dell, M., 279, 280, 919 Perlmann, G. E., 234, 318, 356, 357, 396, o h m , 0. E., 388, 398 489, 490, 517, 518, 524, 636, 648 Ogle, J. D., 41, 137, 224, 318 Perrin, F., 336, 337, 398 Ogston, A. G . , 101, 139 Peruts, M. F., 426, 483, 504, 637,642 Oikawa, A., 486, 641 Peterlin, A., 331, 347, 363, 370, 3g9 O’Konski, C. T., 355, 398 Peters, T., Jr., 277, 278, 280, 281, 282, Olcott, H. S.,234, 318 284, 318 Olivry, G., 187(174), 196 Oncley, J. L., 65, 9G, 136, 329, 332, 334, PeterRon, D. L., 415, 423, 642 Pfleiderer, G., 273, 321 335, 338, 341, 355, 390, 398 Oosterhaan, R. A., 161(89), 199,235,305, Phelps, R. A., 285, 318 Philppoff, W., 328, 381, 399, 430, 6-42 316, 318 Phillips, D. C., 402, 452, 462, 466, 483, Orekhovich, K. I)., 64, 136 504,5OG, 640 Orekhovich, V. N., 41, 64, 65, 67, 69, 70, 91,99, 100, 101, 102, 103, 133, 134, 136 Pickett, E. E., 497, 639 Pierce, J. G., 254, 318 Oster, G., 361, 400 Piez, K. A., 4, 5, 31, 32, 33, 34, 35, 74, Ottewell, R. H., 356, 357, 359, 397 76, 78, 1 0 , 101, 102, 103, 104, 119, Oriellet, L., 303, 320 131, 134, 136, 152(53), 175(53), 176 Overbeek, J. T. G., 350, 351, 397 (53), 192 P Platt, J. R., 415, 423, 639 Pleass, W. B., 108, 136 Page, J., 296, $14 Palade, G. E., 148(24, 25, 26, 27), 149 Plock, R. J., 348, 363, 398 Plotnikova, N . E., 64, 136 (26, 27, 30), 150(26), 151(31), 191 Poilroux, M., 154(65), 156(65), 161(65), PalBus, S., 298, 320 1 92 Parker, A. C., 509, 640 Polson, A., 325, 399 Parker, E. A., 59, 129 Popenoe, E. A., 223, 254, 316, 318 Parks, L. W., 266, 318 Porath, J., 294, 319 Parmentier, G., 246, 320 Poroshin, K. T., 41, 136 Partridge, S. M., 229, 318 Porter, K. R., 360, 399 PasBro, L., 178(147), 180(147), 194
Nielsen, S. O., 431, 637 Niemann, C., 36, 128, 227, 316 Nishigai, M., 64, 134 Nishihara, T., 67, 68, 69, 70, 74, 76, 78, 81, 82, 100, 101, 102, 104, 130, 134 Noda, H., 41, 64, 65, 67, 184 Noguchi, J., 312, 316 Nomoto, M., 291, 318 Nordwig, A., 41, 191 Norland, K. S., 434, 436, 441, 451, 457, 458, 459, 474, 640, 641 North, A. C . T., 3, 43, 44, 45, 48, 51, GO, 61, 62, 129, 130, 136, 136, 483, 504,
557
AUTHOR INDEX
Porter, R. R., 181(152), 194, 285, 519 Pouradier, J., 97, 123, 136 Pratt, M. I., 65, 67, 13.4 Price, F. P., 117, 136 Price, V. E., 235, 316 Probst, W. J., 251, 316 Prusik, Z., 163(100), 193 Pullin, A. D. E., 18, 44, 128 Putnam, F. W., 285, 318
Q Quastel, J. H., 274, 319 Quiocho, F. A., 279, 280, 319
R Rabinowitz, J., 272, 315 Ramachandran, G. N., 47, 48, 51, 52, 53, 54,55,56,57, 109,136,528,642 Ramachandran, L. K., 223, 226, 246, 261, 277, 278, 280, 281, 282, 283,286, 287, 289, 290, 303, 316, 316, 319 Ramakrishnan, C., 55, 56, 133 Randall, A. A , , 21, 24,27, 28,130,461,638 Randall, J. T., 3, 18, 42, 43, 44, 45, 48, 51, 53, 60, 61, 62,93, 94, 109, 116, 129, 130, 133, 136, 136
Bands, D. G., 359, 400 Rasmussen, H., 294, 319 Ravin, H. A., 171(118), 193 Ray, P. M., 245, 319 Ray, W. J., Jr., 270, 319 Reboud, J. P., 146(22), 147(22), 152(60), 191, 19.9 Redfield, R. R., 141(6), 191, 294, 296, 31.4, 319 Reese, C. B., 314, 319 Reichmann, M. E., 359, 369, 397, 399 Reiner, M., 378, 379, 399 Resnick, H., 186(167), 196 Ressler, C., 223, 254, 255, 316, 319 Rice, R. V., 67, 68, 109, 127, 136 Rice, S. A., 473, 642 Rich, A., 12, 13, 14, 16, 25, 43, 48, 49, 50, 52, 53, 54, 150, 138, 447, 528, 657, 642 Richard, E. G., 83, 157 Richards, F. M., 186(170, 171, 172, 173), 187(175, 176, 177), 188(184), 196, 294, 295, 319, 320 Riedel, A., 38, 191
Riese, H. C . A., 123, 124,125, 126, 188 Riley, D. P., 354, 396, 430, 452, 6.96 Riley, G., 234, 919 Riseman, J., 346,363,370,398, 399 Rittel, W., 223, 319 Robbins, K. C., 142(12), 191 Robb-Smith, A. H. T., 5 , 196 Roberts, C. W., 223, 316 Robertson, A. V., 224, 240, 241, 243, 276, 517, 319 Robinson, C., 108, 109, 110, 136,478, 484, 638, 642 Robinson, J. R., 327, 399 Robinson, R. A., 251, 319 Roche, J., 209, 210, 219 Roe, J. H., 147(19), 191 Roig, A., 473, 641 Rollett, R . S., 519, 641 Roscoe, R., 123, 132 Rosen, I. G., 442, 464,465,659 Rosenfeld, V. L., 408, 435, 642 Rosenheck, K., 423, 424, 432, 454, 481, 484, 511, 521, 642 Rossi-Fanelli, A., 280, 319 Rossman, M. G., 483, 504, 6-42 Rothman, S., 329, 400 Rothschild, H. A., 153(61), 192 Rougvie, M. A., 4, 5, 42, 91, 92, 131, 136 Rouse, P. E., Jr., 370, 374, 375, 399 Ilovery, M . , 152(43, 44), 154(43, 44, 65) , 155(67), 156(65, 67), 158(67, 75), 159(79), 161(65), 162(85, 86), 163 (105), 164(105), 165(85), 166(85), 167(85), 170(116), 171(126), 172 (126), 186(169), 193, 193, 194, 196 Itudloff, V., 223, 297, 316 Rupley, J. A., 157(73), 174(132, 133), 192, 194, 497, 527, 641 Ryle, A. P., 184(161, 162), 185(163), 196, 223, 319 S
Sadeh, T., 226, 232, 234, 318 Sadron, C., 332, 337, 339, 368, 390, 599 Saito, N., 363, 399 Sakakibara, S., 41, 134 Salo, T. P., 59, 60, 61, 128 Sanger, F., 184(161, 162), 196, 222, 223, 229, 235, 237, 318, 319 Sarda, L., 143(16, 17), 147(1B), 148(16),
558
AUTHOR INDEX
177(16, 17, 143), 178(145, 146, 147), 179(146),180(146, 147), "148, 149), 191,194
Sasisekharan, V., 8, 14, 16, 19, 26, 55, 56, 57, 133, 136, 136, 417, 642 Satyanarayana Rao, G. J., 279, 280, 319 Saum, A. M., 358, 398 Saunders, P. R., 103, 123, 136, 137 Saunders, W. M., 96, 101, 138 SautiBre, P., 296, 316 Savitz, D., 43b, 515, 648 Saxena, B., 410, 412, 643 Scanu, A., 71, 128 Scatchard, G., 65, 96, 136, 198, 919,329, 355, 398 Schaad, J. A., 59, 120 Schachman, H. K., 83, 137 Schaffer, N. K., 237, 305, 819 Schaffner, A., 175(138), 194 Schellman, C., 73, 104, 136, 403, 417, 439, 463, 464, 491, 499, 500, 510, 642 Schellman, C. G., 403, 457, 485, 491, 502, 503, 508, 519, 642 Schellman, J., 186(165), 196 Schellman, J. A., 6,73, 104, 106, 112,138, 136, 251, 317, 403, 404, 405, 409, 410, 415, 417, 418, 439, 443, 446, 447, 453, 457, 460, 462, 463, 464, 473, 475, 477, 482, 485, 486, 487, 488, 489, 491, 496, 497, 498, 499, 500, 502, 503, 508, 510, 513, 514, 516, 619, 520, 526, 638,639, 641,642
Scheraga, H. A., 97, 116, 131, 138, 325, 337, 339, 340, 343, 346, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 374, 391, 392, 394, 395, 396, 398, 399, 400, 474, 476, 496, 504, 637,639, 642 Scherrer, H., 274, 320 Schleich, H., 33, 131 Schlenk, F., 266, 318 Schleyer, M., 38, 40, 131 Schmidt, E., 251, 319 Schmir, G. L., 226, 252, 256, 257, 258, 259, 260, 261, 262, 263, 264, 272, 316, 319
Schmitt, E. E., 435, 442, 464, 465, 639 Schmitt, F. O., 38, 43, 46, 58, 59, 60, 61, 62, 63, 64,65, 70, 71, 72, 99, 131, 132, 136 Schneider, F., 33, 136
Schonberg, A., 256, 319 Schopf, A., 229, 321 Schofield, K., 314, 319 Schram, E., 283, 319 Schramm, G., 223,285, 286, 287, 314 Schroeder, W. A., 36, 37, 45, 136, 137 Schrohenloher, R. E., 41,137 Schultz, J., 226, 230, 231, 319 Schumaker, V. N., 83, 137 Schuytema, E. C., 41, 137 Schwartz, E. T., 223, 317 Schwyzer, R., 223, 319 Scott, D. B., 72,134 Scott, F. L., 256, 319 Scott, R. A., 504, 642 Scott, R . L., 262, 319 Seeds, W. E., 3, 61, 136, 361, 400 Seibles, T. S.,175(139), 194 Seifter, S., 41, 65, 67, 68, 69, 71, 84, 85, 86, 87, 88, 90, 98, 118, 130, 131, f34, 137 Sekora, A., 58, 133 Sela, M., 15, 20, 21, 22, 24, 25, 26, 27, 28, 29, 74, 77, 106, 107, 138, 133, 137, 226, 230, 231, 294, 310, 314, 321, 457, 458, 461, 462, 474, 514, 526, 639, 642, 643
Seligman, A. M., 171(118), 198 Shalitin, Y., 312, 380 Shaltiel, S., 226, 271, 272, 298, 319 Shapiro, R., 314, 319 Shapiro, S. K., 266, 380 Sharman, L. J., 371, 399 Shaw, D. C., 229, 235, 318, 319 Shay, H., 230, 319 Sheehan, J. T., 236, 280, 316, 316 Sheppard, R. C., 280, 316 Sherman, J., 19, 136 Sherry, S., 152(55), 19.2 Shibnev, V. A., 41, 136 Shin, K. H., 235, 321 Shive, W., 265, 318 Shooter, E. M., 403, 6.49 Shore, V. C., 402, 452, 462, 466, 483, 504, 506, 640 Shore, W. S., 359, 400 Shpikiter, V. O., 41, 65, 67, 68, 70, 91, 99, 100, 101, 102, 103, 133, 134, 136 Shulman, S., 356, 396 Shumaker, J. B., 356, 398
AUTHOR INDEX
Shurman, M. M., 59, 133 Siegel, B. M., 360, 361, 399 Siehr, D. J., 244, 320 Siekevitz, P., 148(24, 25, 26), 149(26, 30), 150(26), 151(31), 191 Sigmund, F., 15, 138 Signer, R., 97, 134, 364, 399 Sikorski, J., 14, 134 Silvester, N. R., 4, 5, 6, 33, 34, 137 Simet, L., 305, 319 Simha, R., 96, 134, 329, 332, 341, 390, 398, 999,400 Simmons, N. S., 325, 346, 355, 361, 362, 396, 421, 422, 423, 434, 435, 490, 492, 493, 503, 505, 507, 529, 530, 531, 642 Simpson, R. B., 359, 398,496,497,640,643 Simpson, W. T., 415, 423, 424, 432, 466, 639, 642, 643 Singh, B. K., 410, 412, 643 Singleton, L., 33, 137 Sinn, L. G., 223, 252, 316 Sinsheimer, R. L., 83, 137 Sittel, K., 375, 399 Slobodian, E., 225, 318, 3.20 Smith, B. W., 147(19), 191 Smith, C. R., 108, 109, 113, 114, 117, 119, 197 Smith, E. L., 162(88), 176(141), 193, 194, 223, 317, 318, 423, 638 Smith, L. F., 184(162),196, 223, 319 Smoluchowski, M. v., 336, 351, 399 Smyth, D. G., 293, 295, 320 Snell, N. S., 280, 318 Snyder, H. R., 228, 320 Sgirensen, M., 201, 819 Sorensen, S. P. L., 201, 202, 219 Sokal, Z., 4, 5, 131 Sokolvsky, M., 226, 232, 234, 318 Sones, R. H., 371, 399 S&m, F., 163(94, 95, 96, 97, 98, 99, 100, 101), 193, 235, 280, 317, 320 Southgate, H., 209, 211, ,918 Spach, G., 430, 641 Spaokman, D. H., 161(83), 162(83), 182 (157), 183(157), 185(157), 189(157), 193, 196, 269, 293, 300, 320 Springell, P. H., 287, 320 Spurlin, H. M., 328, 399 Stahmann, M., 463, 636 Stainsby, G., 97, 123, 189, 137
559
Stake, M., 435,442,464,465,639 Stanley, W. M., 223, 289, 317, 3.20 Stark, G. R., 188(183), 196 Staudinger, H., 330, 387, 399 Stauff, J., 486, 514, 515, 643 Steber, A., 98, 131 Steere, R. L., 361, 399, 400 Stein, W. D., 188(181, 182), 196 Stein, W. H., 149(33), 161(83), 162(83), 181(153), 182(157, 158, 160), 183(157), 185(157), 188(180, 183), 189(157), 191, 193, 194, 196, 223, 233, 265, 266, 267, 268, 269, 293, 294, 295, 300, 312, 317, 318, 320 Steinberg, I. Z., 18, 20, 21, 22, 24, 25, 26, 27, 28, 106, 107, 137, 403, 457, 458, 460, 461, 473, 474, 640, 642, 643 Steiner, R. F., 359, 397, 527, 643 Sterman, M. D., 497, 643 Steven, F. S., 497, 643 Stevenson, G., 256, 317 Stewart, C . P., 274, 319 Stewart, J. A., 303, 320 Stiasny, E., 105, 137 Stieglitz, J., 240, 320 Stirling, C. J. M., 238, 256, 320 Stockell, A., 176(141), 194 Stockmayer, W. H., 349, 399, 400 Stokes, A. R., 44, 137, 361, 400 Stokes, R. H., 251, 319 Stracher, A., 106, 137, 514, 643 Strandberg, B. E., 402,452,462,466,483, 504,506,640 Strauss, U. P., 350, 397 Street, A., 427, 636 Streeter, D. J., 387, 399 Stryer, L., 450, 529, 636, 643 Sugits, M., 363, 399 Sund, H., 486, 643 Sturtevant, J. M., 171(122), 194 Sutherland, G. B. B. M., 12, 18, 47, 48, 56, 128, 137 Sutherland, G. L., 265, 318 Svedberg, T., 336, 337, 356, 391, 399 Swan, J. M., 223, 296, 316, 320 Swan, J. N., 160(82), 192 Swanson, S. A., 359, 400 Swindells, J. F., 381, 399 Synge, R. L. M., 225, 280, 380 Szent-Gyorgyi, A., 637
560
AUTHOR INDEX
Szent-Gyorgyi, A. G., 7,77,137,422,423, 434, 435, 467, 487, 490, 492, 493, 503, 505,507, 529, 631,646
T Taborsky, G., 181(156), 190(156), 194 Tabroff, W., 36, 37, 45, 1% Taggart, V. G., 488, 493, 495, 508, 520, 521, 645
Takagi, T., 527, 643 Takahashi, T., 77, 137 Takeda, M., 387, 388, 899 Tallan, H. H., 169(114), 193 Tan, B. H., 514, 640 Tan, W., 162(84), 193 Tanaka, J . , 424, 432, 474, 477, 510, 689 Tanaka, T., 77, 137 Tanford, C., 186(168), 196, 325, 334, 340, 351, 352, 356, 358, 359, 383, 596, 399,
400, 482, 483, 488, 493, 495, 496, 508, 520, 521, 526, 645 Tanner, K . N., 18, 47, 48, 56, 128, 137 Tarbell, D. S., 243, 517 Taylor, D. B., 209,211, 212, 218 Taylor, J. F., 218, 619 Telka, M., 142(11), 175(139), 191, 194, 310, 560 Teller, E., 91, 129
Tennent, H. G., 328,399 Thathachari, Y. T., 55, 56, 57,153, 136 Thiele, E. H., 142(10), 191 Thimann, K. V., 245, 319 Thomas, C . A., Jr., 83, 157 Thompson, E. 0. P., 174(130), 194, 222, 229, 237, 294, 319, 3.90 Tietre, F., 312, 380 Tinoco, I., Jr., 416, 424, 430, 432, 443, 448, 452,466, 648 Todd, A,, 314, 519, 403, 421, 485, 509, 643 Todd, A. R., 314, 316 Toennies, G., 265, 268, 320 Tom&Bek, V., 163(99, 101), 193, 235, 320 Tomlin, S. G., 59, 137 Traumann, K., 226, 233, 321 Tresser, P., 105, 157 Trigny, I,., 123, 136 Trippett, S., 223, 254, 916, 319 Tristram, G. R., 31, 32, 137, 320, 497, G43 Tritch, H., 294, 310, 314 Trommel, J., 453, 643 Trotter, I. F., 425, 427, 452, 656, 637
Trueblood, K. N., 8, 9, 11, 16, 130 Trupin, K. M., 181(154), 190(154), 194 Tsao, Tien-Chin, 65, 67, 136 Tsuboi, M., 477, 478, 479, 519, 643 Tsugita, A., 223, 520 Tsuruta, E., 387, 399 Tuijnman, C. A. F., 382, & . I 0 Tunnicliffe, H. E., 274, 319 Tuppy, H., 223, 237, 298, 320 Turnbull, J. H., 234, 319 Turner, J. E., 513, 515, 6.43 Tiistanovskii, A . A , , 64, 136
U Uhlig, H . , 223, 285, 286, 287, 514 Ulmer, D. D., 486, 516, 530, 643 Umstatter, H., 388, 400 Umes, P. J., 432, 433, 435, 436, 453, 454, 457, 466, 474, 484, 488, 503, 524, 527, 530, 532, 533, 643
TJzmnn, I,. TJ.,484, 645
V Vajda, T., 226, 232, 320 Vallee, B. L., 174(132, 133, 134, 135, 13A), 194, 486, 516,530, G4S
Van Bommel, A. J., 16, 188 Vand, V., 43, 44, 129, 425, 637 Vanderhaeghe, H., 246, 360 VanBEek, J., 163(94, 96, 97, 98, 100, 101), 195
Van Lancker, J. L., 151(39), 191 Van Rotterdam, J., 161(89), 193 Veis, A., 96, 97, 100, 107, 109, 127, 167 Venet, A. M., 97, 123, 156 Vincent, J. M., 430, 641 Vinograd, J. R . , 123, 124,125,126,128 Viswanatha, T., 169(112, 113), 171(123), 193, 194, 224, 280, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 320 Vithayathil, P. F., 295, 319 Vithayathil, P. J., 186(173), 187(175, 176, 177), 188(184), 196, 294, 320 von Braun, J., 270, 320 von Hippel, P. H., 41, 67, 68, 69, 77, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 104, 105, 108, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 126, 132, 157
561
AUTHOR INDEX
W Wada, A., 355, 397, 404, 430, 431, 435, 446, 450, 473, 474, 476, 477, 478, 479, 498, 519, 637, 642, 643 Wagner, M. L., 357, 400 Waldsohmidt -Leite, E., 175(138), 194 Waley, S. G., 15, 132, 157 Walter, J., 405, 638 Ward, A. G., 95, 103, 123, 133, 136, 137 Wardlaw, A. C., 223,316 Warren, W. J., 59, 129 Watson, H. C., 402, 452, 462, 466, 483, 504, 506, 640 Watson, J., 15, 132 Watson, M. R., 4, 5, 6, 33, 34, 157 Watson, M. T., 359,397, 497,658 Waugh, D. F., 357, 400 Weaver, E. S., 81, 108, Ill, 113,114,117, 118, 126, 130 Weber, E., 223,285,286,287,314 Weber, G., 359, 400 Weber, R. E., 186(168), 196, 496, 526, 643
Wegemer, N. J., 325, 371, 398 Weil, L., 142(11), 175(138, 139), 191, 194, 280, 305, 310, 320 Weir, C. E., 75, 137 Weiss, E., 100, 101, 102, 136 Weiss, K. W., 280, 316 Weiss, S. B., 149(29), 191 Weissberg, S. G., 329, 400 Welsh, H. K., 7, 8, 11, 134 Werner, B., 140(3), 191 Wessely, F., 15, 138 West, R. W., 254, 314 Wetlaufer, D. B., 422, 423, 434, 435, 490, 492, 493, 503, 505, 507, 529, 531, 642 White, E. H., 274, 520 White, F. H., Jr., 181(151), 189(185, 187, 189), !94, 196, 223, 316, 320,489, 514, 6@ Whiteley, M. A., 254, 314 Whitfield, P. R., 314, 321 Wiberg, E., 257, 316 Wiederhorn, N. W., 75, 158 Wieland, Th., 229, 235, 273, 321 Wiener, E., 298, 318 Wienhoven, J. F., 105, 124, 125, 133 Wilcox, P. E., 162(84), 193 Wilkins, M. H. F., 361, 400
Wilkinson, G. R., 3, 18, 53, 61, 93, 94, 109, 116, 189, 136 Will, G., 483, 504, 6.42 Williams, A. P., 33, 34, 158 Williams, D. E., 142(9), 191 Williams, J. W., 65, 96, 101, 136, 158 Williams, N. J., 280, 316, 516 Williams, R. C., 361, 400 Wilson, J. G., 223, 226,291, 293, SI6 Wilson, J. N., 9, 10, 132 Wilson, W., 234, 319 Winkler, M., 519, 530, 643 Winita, M., 224, 225, 236, 317, 321 Winstein, S., 238, 256, 316, $19 Wirtz, K., 535, 643 Witkop, B., 171(123), 19.6, 222, 223, 224, 225, 226, 235, 240, 241, 243, 244, 245, 246, 247, 248, 250, 251, 252, 256, 260, 261, 267, 268, 269, 271, 272, 274, 275, 276, 277, 278, 280, 281, 282, 283, 286, 287, 289, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304, 314, 316, 316, 517,318, 310, 320, 3.81 Wittmann, H. G., 287, 321 Wittmann-Liebold, B., 223, 297, 316 Wohlisch, E., 79, 158 Wolf, K., 33, 131, 138 Wolpers, C., 58, 138 Wong, K. Y., 119, 120, 126, 137 Wong, R. C., 169(112), 193 Wood, D. L., 18,47, 48,56, 1.88, I37 Wood, G. C., 72, 138 Wood, H. N., 305, 307, 581 Woods, H. J., 427, 636 Woodward, A. E., 434, 465, 478, 642 Woody, R. W., 416, 448, 452, 643 Wooton, J. F., 307, 321 Wootton, J. F., 169(111), 193 Worthington, C. R., 59, 137 Wright, B. A., 75, 138 Wilre, H., 513, 515, 643 Wunsch, E., 41, 131 Wyckoff, R. W. G., 59, 61, 138 Wyman, J., 198, 918, 325, 400
Y Yajima, H., 223, 317 Yamakawa, H., 348, 398, 400 Yltmasaki, M., 173(129), 194 Yamashina, I., 152(56, 57), 168(57), 193 Yanaihara, N., 223, 317
562
AUTHOR INDEX
Yang, J. T., 73, 108, 138, 186(166), 196, 325, 345, 350, 354, 355, 359, 362, 364, 365, 366, 367, 368, 369, 371, 372, 373, 395, 397, 398, 400,403, 404, 409, 410, 412, 414, 415, 417, 418, 423, 430, 431, 432, 434, 437, 442, 446, 449, 450, 467, 472, 473, 474, 476, 477, 478, 479, 482, 486, 487, 488, 489, 493, 494, 497, 498, 500, 501, 508, 513, 521, 636, 637,641, 644
Yaron, A., 30, 138, 226, 230, 231, 361 Yoshida, A., 154(65), 156(65), 161(65), 196, 487, 527, 639 Young, E. G.,67, 68, 138 Young, J., 223, 360 Young, J. Z., 31, 138
Yphantis, D., 465, 639 Yphantis, D. A., 357, 400 Z
Zachariades, P. A., 64, 138 Zahn, H., 226, 228, 233, 236, 361 Zervas, L., 236, 321 Ziegler, F., 175(138), f94 Zimm, B. H., 112, 138, 332, 346, 348, 349, 362, 370, 397, 400, 465, 473, 474, 475, 476, 504, 644 Zuber, H., 223, 310 Zurn, L., 225, 226, 227, 228, 236, 361 Zussman, J., 8, 138 Zwanzig, R. W., 348, 398
Subject Index A Acinar cells, pancreatic, enzyme synthesis in, 148-153 Adrenocorticotropin, primary sequence of, 223 Alanine polypeptide, @-form,479 conformation in solvents, 44&441 helix-coil transition, 474, 476, 477 optical rotation, 434, 449, 450, 452 Alcohol dehydrogenase, Cotton effects of, 530 optical rotation, 486 Allohydroxylysylglycinamide, 228 Amino acid [s) , functionality of, 224 NBS effect on, 263 possessing functional groups, 224 y-Aminobutyryl peptides, cleavage of, 273-275 Ammonium sulfate, use in salting-out, 216 Amylase ( 8 ) , chromatography of, 143 optical rotation, 486 Antibiotics, 345440 Arachin, optical rotation, 486 Aspartic acid, in peptide cleavage, 226 from proteins, 229 Aspartic acid polypeptide, film, optical rotation, 455456 optical rotation of, 434, 451, 452, 472 preferential cleavage of, 231 Aspergillus saitoi, trypsinogen activator from, 168
B Baikiain amide, 242 Bence-Jones proteins, 518 optical rotation, 491 Butyric acid, poly-DL-a-amino-n-, optical rotation, 449 Byssus threads, as collagen, 6
C Calcium chloride, effect on polyproline, 25 Capillary viscometer, 375-378 corrections for, 379-384 design of, 385-387 Carbamoyl choline, 153 Carboxyhemoglobin, salting out of, 199, 200, 201-202 Carboxymyoglobin, salting out of, 200, 205-208 Carboxypeptidase ( 8 ) , amino acid composition of, 176 optical rotation, 486 Carboxypeptidase A, bovine, 173-174 role of metals in, 174 Carboxypeptidase B, bovine, 175-176 porcine, 175176 Casein, optical rotation and structure, 490, 517 Catalase, Cotton effects and, 515 Cellulose chromatography, of pancreatic juice, 141-148 Cellulose trinitrate, viscosity of, 325 Cephalosporin C, cleavage of, 274-275 Chymotrypsin, denatured, optical rotation, 497 folding in, 499, 500 helical content, 511 methionine role in, 270 optical rotation, 487 tertiary structure, 526 three chains of, 158-159 Chymotrypsinogen(s), amino acid composition, 162 bonds of, 158 bovine, 154-163 denatured, optical rotation, 497 folding in, 499, 500 “free tail,” 156 helical content, 511 NBS effect on, 307
663
564
SUBJECT INDEX
optical rotation, 487 disulfide bonds and, 515 pancreatic activation of, 146 peptides of, 163 structural analysia of, 484, 527 Chymotrypsinogen A, chromatography of, 141-146, 166 inactive precursor of, 152 nomenclature of, 154 pancreatic, biosynthesis of, 149-150 porcine pancreatic, 165-167 primary structure of, 161-163 terminal structure of, 154-156 treatment with sulfite, 160-161 Chymotrypsinogen B, bovine, 163-164 chromatography of, 143, 104 inactive precursor of, 152 nomenclature of, 154 Clupein, optical rotation and structure, 106, 443, 446, 487 Coaxial cylindrical viscometer, 378-379 Collagen, acetylation of, 77 aggregation of, 62 amino acids of, 2-3, 4, 30-35 calfskin, 32 chemical characteristics of, 4 collagenase effect on, 84 enzyme studies on, 83-91 “equivalent scattering groups” in, 45 fibrils, electron micrography of, 43, 59 histology of, 4 structure of, 58-64 X-ray pattern of, 2, 4, 5 fish, 31 -gelatin transformation, 3, 75-83 “heat precipitation” of, 71 human tendon, 32 hydration of, 93-94 hydrogen bonding in, 94 hydroxyproline in, 2, 4, 5, 30 imino acid content of, 76 interchain linkages in, 39 invertebrate, 33-35 iodination of, 71-72 length of, 346 linkages of, 99 optical rotation and structure, 72, 498, 528-529
ox bone, 32 oxskin, 32 partial regeneration from gelatin, 109 peptides from, 3 5 4 1 physical characteristics of, 4 physicochemistry of, 3 polypeptide backbones of, 50-51 pro-, 99 rat skin, 32 reviews on, 3 “secreted,” 5-6 sheep tendon, 32 side chain position in, 52 soluble, isolation of, 64-75 physicochemical properties of, 06-07 sonicated, 70 structure, i n solid state, 42-64 three-chain, 47 water in, 91-94 sugars associated with, 33, 35 swin bladder, 32 synthetic polypeptides related to, 6-30 tropo-, 63, 68, 99 tryptic digestion of, peptides from, 40 types of, 5-6 vertebrate, 31-33 X-ray studies of, 42 Collagenme, 41 effect on collagen, 64, 84 specificity, 89-91 Cornein, as collagen, 5 Cotton effects, 529-534 heme groups and, 515 in optical rotation, 419-424 Creatine phosphokinase, NBS treatment of, 279 Cysteine, methionine-, polypeptide, helix-coil transition, 474, 476 in peptide cleavage, 226 peptides, sulfonium derivatives of, 234 Cysteine diketopiperasine, hydrolysis of, 228 Cytochrome c, cleavage of histidine peptide of, 297298 Cotton effects, 515
565
SUBJECT INDEX
D
G
Dehydrogenases, Cotton effects of, 515- Gelatin (s), a-,and &components, 102 516 amino acid composition of, 30-35 Dehydropeptides, i n protein cleavage, annelida cuticle, 34 232-235 carpskin, 32 Denaturation, chicken tendon, 32 effect on proteolysis, 86 codskin, 32 viscosity and, 389 coelenterate, 34 Deoxyribonuclease, -collagen transition, 75-83 chromatography of, 143 collagenase study of, 127 optical rotation, 487 commercial preparation of, 95-96 Drude equation, 406-407, 534 crocodileskin, 32 de-esterified, mutorotation, 119 E DMF effect on, 107 Edestin, optical rotation, disulfide echinoderm, 34 bonds and, 513 formic acid effect on, 107-108 Elastase, 142 gelation mechanism of, 125-127 optical rotation, 490 gels, Elastin, as noncollagen, 6 optical rotation of, 125 Elastoidin, as collagen, 5 properties of, 122-127 Enterokinase, as trypsin activator, 152 salt-containing, 124 Enzyme (s), linkages of, 98 fractionation by salting out, 197-219 low temperature properties, 108127 pancreatic, see under Pancreatic lungfish skin, 32 proteolytic, cell protection against, mollusca body wall, 32 151-152 mutorotation of, 113-122 Equivalent ellipsoid, length estiinrttion optical rotatory properties of, 74, 103of, 344-345 107, 491, 529 Esterase(s), active site study of, 235 partial regeneration to collagen, lo!! peptides from, 37 F pigskin, 32 python skin, 32 Fetuin, optical rotation, 487 sharkskin, 32 Fibrinogen, size and shape, 96-103 length of, 346 from soluble collagen, 96-103 optical rotation, 487 solvent effects on, 107-108 physical properties, 355, 359-361 structure of, 95-127 salting out, 200 sugars associated with, 33 viscosity of,360-361 toadskin, 32 Fibroin(s), whaleskin, 32 cleavage of, 225 Globin@), optical rotation, 487 helical content, 501-502 Globin-H, helical content, 509, 510 optical rotation of, 449 Globin-M, helical content, 509, 510 Fibrous proteins, structure of, 55 @-Globulin, optical rotation, 487 Ficin, optical rotation, 487 Flexible coils, theories for, 346-349, 369- yGlobulin, denatured, optical rotation, 497 379 folding in, 500 Flow birefringence, non-Newtonian viscosity and, 368-369 helical content, 511 Follicle-stimulating hormone, 487 NBS effect on, 285
566
SUBJECT INDEX
optical rotation and structure, 491, 518, 519 Glucagon, enzyme cleavage of, 252 optical rotation, 487 primary sequence of, 223 tryptophan oxidation in, 251 tryptophyl bond nlen.vaPe in. 261, 281 Glutamic acid, lysine-, polypeptide, helix-coil transition, 476, 494-495 optical rotation, 446, 470-474, 498499 i n peptide cleavage, 226 polypeptide, @-form,481 cleavage of, 273-275 conformation study of, 432 Cotton effects, 529, 531, 532, 533 film, optical rotation, 455 helical content, 502,504,505,509-510 helix-coil transition, 474, 477 light scattering of, 431 folding in, 500-501 optical rotation of, 404, 417, 422, 434-444, 445-446, 450, 464-465, 498 random coils, optical rotation, 440 tyrosine copolymer, Cotton effects of, 531 helical content, 531 helix-coil transition, 474 optical rotation of, 442,443,446, 453, 454 Glutamic acid dehydrogenase, optical rotation, 487 Glyceraldehyde phosphate dehydrogenase, optical rotation, 488 GIycine, bond angles of, 8 in collagen, 2, 4, 30, 35 crystal structure of, 7 frequency in collagen peptides, 36 polypeptide, 49 optical rotation, 415, 447 properties of, 11-14 structure of, 12-13 proline copolymers, 28-30 a-Glycoprotein, optical rotation, 516 Glycylglycine, stereochemistry of, 9-10 Glyoxylase, structure, 484
Gorgonin, as collagen, 5 Gramicidin, cleavage of, 289-291 NBS cleavage of, 290-291 purification of, 292
H Heme group, effect on optical rotation, 515 Hemoglobin(s), 01- and &chains of, 297 Cotton effects and, 515 denatured, optical rotation, 503,504 NBS effect on, 282-283 primary sequence of, 223 salting out, 210 structure, 483, 484 Histidine, i n peptide cleavage, 226, 270-273 polypeptide, helix-coil transition, 474 unusual rotatory properties of, 458, 459-460 Histone, helical content, 509, 510 optical rotation, 488 Homoserine lactone, hydrolysis of, 228 Hydrodynamic ellipsoid, concept of equivalent, 33&344 Hydroxy acid amides, hydrolysis of, 227 Hydroxyamino acids, 224 Hydroxylsine, i n collagen, 4 in embryonic muscle, 31 i n peptide cleavage, 226 in spongin, 35 Hydroxyproline, bond angles of, 8-9 i n collagen, 2, 4, 5, 30 crystal structure of, 8-9 in nemocyst protein, 31 i n peptide cleavage, 221 poly-0-acetyl-, 21, 27 polypeptide, 26-28 unusual rotatory properties of, 458, 461-462 0-tosyl-, peptide cleavage of, 275-277
I Ichthyocol, 67-69 as collagen, 5
667
SUBJECT INDEX
collagenase effect on, 84-86 molecular weight, 101 specific rotation, 115 Indoleacetic acid, NBS effect on, 244 Indolepropionic acid, transformation of, 245 Infrared spectroscopy, of synthetic polypeptides, 428-429 I-peptide, tryptophyl bond cleavage of, 281 Insulin, denatured, optical rotation, 497,498 folding in, 499, 500 helical content, 501-502,509-511 helix-coil transitions, 494 optical rotation, 482, 488, 494 oxidized, optical rotation of, 443, 446447 primary sequence of, 223 structure, 484, 519-520 tyrosine cleavage in, 293-294 Insulinase, 142
Luteinizing hormone, optical rotation and structure, 490, 518 Lysine, carboxyl activation of, 236 Lysine polypeptide, 8-form, 481 conformational study of, 432 helical content, 502, 504, 509 polypeptide, helix-coil transition, 473, 474 optical rotation and structure, 430431, 434, 435, 445-446, 463-464, 468-469, 470-471, 472, 498, 501 Lysoayme, denatured, optical rotation, 497 folding in, 500 helical content, 509, 510, 511 NBS effect on, 282 optical rotation, 488 selective cleavage of, 251 tertiary structure, 526, 527 tryptophyl bond cleavage in, 281
L
Macroglobulins, optical rotation, 491 Macromolecules, viscosity of, 323-400 Magnesium sulfate, use in salting out, 216-217 Malic dehydrogenase, optical rotation, 488 Melanocyte-stimulating hormone, primary sequence of, 223 Mercaptalbumin, NBS effect on, 282 optical rotation, 488 Meromyosin, optical rotation, 492 Methionine, i n peptide cleavage, 226 polypeptides, cleavage of, 265 helix-coil transition of, 474, 476 optical rotation of, 422, 435 S-adenosyl-, 265 8-Methylene oxindole, 245 Moffitt equation, 413-418 for mixtures of helices and random Coils, 462472 Muscle, embryonic, hydroxylysine in, 31 Myeloma globulins, optical rotation and structure, 491, 516, 518 Myoglobin, conformation of, 452453
Lactic acid dehydrogenase, optical rotation, 488 P-Lactoglobulin, folding in, 499, 500 helical content, 510, 511 helix-coil transformations, 495-496 optical rotation and structure, 482, 488, 491, 516-517, 52&522 Lactonization, 245-246 Leucine polypeptide, helix-coil transition, 474, 476 optical rotation, 416, 433434, 435, 449 Leucyl-L-prolylglycine, stereochemistry of, 9-10 Light scattering, of synthetic polypeptides, 429-430 Lip ase , chromatography of, 143 porcine, 176-181 activity of, 178-181 Lithium bromide, effect on gelatin rotation, 105 effect on polyproline, 25-26 Liver, enzymes, chromatography of, 145 proteins, salting out of, 212
M
568
SUBJECT INDEX
Cotton effects on, 515 crystallography of, 402 denatured, optical rotation, 503 helical content, 511, 512 optical rotation of, 453-454,466,488 X-ray structure, 483 Myosin, length of, 346 optical rotation, 492 para-, optical rotation, 422 tropo-, optical rotation, 422 tryptic digestion of, 87
N Newtonian viscosities, theories for rigid particles 0
Optical rotation, of helical structures, 412-418 of proteins and peptides, 402-544 Ornithine, carboxyl activation of, 236 Ovalbumin, denatured, optical rotation, 497 folding in, 499, 500 helical content, 509, 510, 511 helix-coil transformation, 495-496 optical rotation, 482, 488 disulfide bonds, 513 salting out, 201 tryptophyl bond cleavage in, 281 Ovomucoid, optical rotation, 490, 516 Oxindole-3-acetic acid, 244 Oxytocin, bromine cleavage of, 255 primary sequence of, 223
P Pancreas, acinar cells, enzyme biosynthesis in, 148-153 exocrine, proteins of, 139-195 Pancreatic enzymes, biosynthesis of, 141-153 nomenclature of, 154 Pancreaozymin, action of, 140 Pankrin, 142 Papain, optical rotation, 488 primary sequence of, 223
Paramyosin, denaturation, 490 helical content, 511 optical rotation, 492 Particle length, determination of, 364365 Penicillium janthinetlum, trypsinogen activator from, 168 Pepsin, folding in, 500 helical content, 509, 510 optical rotation and structure, 490, 517-518 Pepsinogen, optical rotation and structure, 489, 518 Peptidase(s), active site studies on, 238 Periodic acid, use in cleavage studies, 314 Phalloidine, lactone of, 229 Phenylalanine, glutamic acid polyppptide, optical rotation of, 478 Phloretic acid, brominatiou of, 252--253 Phloretylglycine, cleavage of, 264 oxidation of, 256-262 Phosphoglucomutase, methioninr! role in, 270 Phosvitin, dephosphorylation of, 234 optical rotation, 490 Pinna nobilis, t ropomyosin , conformation, 492-494 helical content, 504 Plasma, salting out of, 211 Poly-8-benzyl-L-aspartate, helix-coil transitions, 472, 474, 476, 477 Poly -y -benzyl-L-glu tamate conformation, in solvents, 142 Cotton effect and, 421 Drude equation and, 417 helix-coil transitions, 472, 473-474 length of, 346 light scattering of, 429430 meso-helix, rotation of, 416 optical rotation of, 403404, 422, 443ff. viscosity of, 324-325, 353, 365-368 Poly-ymethyl-L-glut amate, &pattern of, 428, 479, 480
X-ray diffraction of, 425-426
569
SUBJECT INDEX
Polypeptides, &conformation, optical rotation of, 477-481 chain conformation, 425-432 D-, optical rotation of, 450 films, optical rotation of, 455-456 helical, extinction coefficient, 423 Moffitt parameters, 432438 optical rotation and, 438-450 helix-coil transitions in, 472477 mesa-helix, optical rotation, 447 mixtures of helices and random coils, Moffitt equation for, 462-472 optical rotation, 401-544 helical vs. randomly coiled, 447 similarity to proteins, 485-504 synthetic, infrared spectroscopy of, 428-429 light scattering of, 429430 optical rotation of, 424-481 of unusual rotatory properties, 456462 viscosity of, 352-355 Polystyrenes, viscosity of, 324-325, 387 Polyvinyl chloride, viscosity of, 387-388 Potassium phosphate, use in salting out, 217 Procarboxypeptidase, inactive precursor of, 152 Procarboxypeptidase A, activation of, 173-174 bovine, 173-174 chromatography of, 143 in pancreatic juice, 144 Procarboxypeptidase B, chromatography of, 143 inactive precursor of, 152 in pancreatic juice, 144 Procollagen, see under Collagen Prolactin, optical rotation, 489 Proline, bond angles of, 8 in collagen, 2, 4, 30 glycine polymers of, 28-30 in peptide cleavage, 226 polypeptides, 14-26, 106-107 hydrodynamic properties, 23-25 infrared spectra of, 17-18 mutorotation of, 18-23
neutral salts and, 25-26 in solid state, 15-18 in solution, 18-26 structure of, 17 unusual rotatory attributes, 456-461 X-ray diffraction of, 16 role in protein configuration, 6-7 sarcosine copolymers of, 30 X-ray diffraction of, 7-8 Protaminase, 142 Protein(s), denatured, optical rotation, 490-491, 503 rotation changes after, 496-499 fibrous, optical rotation, 491-494 X-ray diffraction of, 427 fractionation theory of, 211-217 globular, folding in, 499-501 helices in, 483-485 structure of, 482-483 helical content from optical rotation, 509-513 helix-coil transformations, 494-496 modification of, 221-321 optical rotation, 401-544 deviations from helix-coil pattern, 516-525 disulfide bonds, 513-516 helix us. random coil, 446 reviews of, 403 tertiary structure and, 525-528 prosthetic groups, optical rotation and, 513 scales of helical content, 504-509 selective cleavage of, 221-321 solubility i n salt solution, 198 tryptophan titration of, 280 viscosity of, 325 Pseudoglobulin, salting out of, 200
R Rennin, optical rotation, 490 Reticulin, as collagen, 5 Ribonuclease(s), A, chromatography of, 141-146 bovine pancreatic, 181-190 inactivation of, 188 reaction with iodoacetate, 187-188
570
SUBJECT INDEX
reduction of, 189-190 structure, 183 structure-activity relationship, 185190 terminal sequences of, 187 chromatography of, 143 denatured, optical rotation, 497 folding in, 499, 500 helical content, 501-502, 509-511 helix-coil transitions, 494 methionyl bond cleavage in, 294-297 mouse, 190 NBS cleavage of,293 optical rotation, 489 disulfide bonds and, 513, 514 oxidized, rotation of, 106 primary sequence of, 223 sheep pancreatic, 190 structure, 514 subtilisin-modified, 295-296 tertiary structure, 526 tyrosine cleavage in, 291-294
S Salmine sulfate, optical rotation, 489 Salting out, choice of salt in, 216-217 enzyme fractionation by, 197-219 mixtures of proteins, 207-210 temperature effects on, 217-218 theory of, 198-210 Sarcosine, proline copolymers of, 30 Sea cucumber, “ejected filaments” of, 6 Secretin, 152 action of, 140 Serine, in peptide cleavage, 226 polypeptide, @-form,479, 481 optical rotation of, 443 unusual rotatory properties, 458, 460 Serum albumin, in acid media, 359 @-form,480 6-function of, 357-358 bovine optical rotation and structure, 522-525 conformation in urea, 358 disulfide bonds and optical rotation, 513
folding in, 499, 500 helical content, 501-502, 508-511 helix-coil transformation, 494 optical rotation of, 404, 486, 517 physical properties of, 355-359 salting out of, 200, 207, 210 tertiary structure, 526 tryptophan bond cleavage of, 281,284285 Silk, optical rotation, 491 Sodium sulfate, use in salting out, 216 Sodium thiocyanate, effect on polyproline, 25-26 Somatotropin, optical rotation, 489 Spongin, amino acid composition of, 34 as collagen, 5 hydroxylysine in, 35 Sugars, associated with collagen and gelatin, 33, 35 S-Sulfochymotrypsinogen, 161 trypsin effect on, 163 S-Sulfotrypsinogen, 161
T Tartaric acid, Cotton effect and, 421 Threonine, in peptide cleavage, 226 Thymglobulin, optical rotation, 489 structure, 527 Tobacco mosaic virus, Cotton effects on, 530 hydrodynamic lengths, 369 length of, 346 NBS cleavage of, 288 optical rotation of, 421 physical properties of, 355, 361-363 primary sequence of, 223, 286 selective cleavage of, 251 tryptophan groups in, 285-289 tryptophyl bond cleavage of, 281 viscosity of, 324 Tosyl-L-prolyl-L-hydroxpprolinemonohydrate, stereochemistry of, l(t11 Tropocollagen, see under Collagen Tropomyosin, conformation, 491 helical content, 509, 510 optical rotation, 492
571
SUBJECT INDEX
Trypsin, action of, 311 amino acid composition, 300 bovine pancreatic, 167-171 chromatography of, 169-171 enterokinase, as activator, 152 helical content, 511 NBS effect on, 302-307 oxidized, amino acid composition of, 300
protein cleavage by, 310 -trypsinogen, oxidation of, 298 Trypsin inhibitor, optical rotation, 489 ,
-glutamic acid polypeptide, optical rotation of, 433-436 in peptide cleavage, 226 polypeptide, film, optical rotation of, 456 helix-coil transition, 474 NBS cleavage of, 262 selective cleavage of, 252-265 unusual optical rotatory properties, 456-457, 459
U Ubbelohde viscometer, 385-387
490
Trypsinogen, acetyl -, activation of, 169 NBS effect on, 302 activation of, 167-168 amino acid composition, 172 bonds of, 150 bovine pancreatic, 167-171 chromatography of, 143, 166, 169-171 optical rotation, 489 disulfide bonds and, 513, 515 in pancreatic juice, 144 peptides of, 163 porcine pancreatic, 171-173 precursor of, 132 structural analysis, 527 Tryptophan, carbobenzyloxy-, 246 in lactonization, 246 in peptide cleavage, 226 polypeptides , amino acids liberated from, 250 helix-coil transition, 474 selective cleavage of, 244-252 unusual rotatory attributes, 457-458 titration of, 277-283 Tyrocidin(s), cleavage of, 290 Tyrosine, -histidine competition of, 263
V Valine, methionine-, polypeptide, helix-coil transition, 474 polypeptide, p-form, 481 Vasopressin, bromine cleavage of, 255 primary sequence of, 223 Viscometers, types of, 375-379 Viscosity, complex, 373-375 definition of, 326 denaturation and, 389 electroviscous effect, 351-352 experimental methods of, 3 7 5 4--M extrapolation to zero rate of shear, 384385
of extremely dilute solutions, 387-388 intrinsic, determination of, 328-329 M.W. relationship, 329-330 particle shape from, 333-336 of macromolecules, 323400 Newtonian, 331-336 non-Newtonian, 363-375 polyelectrolytes and, 349-352 power law of, 372-373 of synthetic polypeptides, 352-355 terminology of, 327 Vitrosin, as collagen, 5
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