T H E REVERSIBILITY OF PROTEIN COAGULATION BY M . L. AXSOX A N D A . E. MIRSKY
Coagulation is the most striking reaction of the proteins. For ages it has been a familiar phenomenon because of the prominent rBle it plays in cookery. The change occurring in egg-white when an egg is immersed in boiling water shows how conspicuous protein coagulation can be. Indeed, with combustion and fermentation, protein coagulation was one of the first chemical processes recognized by man. It was only natural, therefore, that this process should early have attracted those interested in the chemical study of living phenomena. For several hundred years the recognition in plant and animal tissues of what is now called protein depended on its property of coagulating when heated. The result of this was that during the nineteenth century there was widespread among biologists and biochemists an acquaintance with even some of the minor details of protein coagulation. More recently, however, there has been a tendency among biologists to neglect the study of protein coagulation, for it has been supposed that coagulation is a degradation process, that coagulated protein is an early stage in the disintegration of the protein molecule, and finally that coagulation, being irreversible, can be of little physiological interest. Investigation of the acid-base properties of proteins appeared to be of greater physiological significance, so that many physiologists are now familiar with protein properties that can be detected only with refined methods, while they are hardly acquainted with some of the gross, easily-recognizable properties. For the investigator of biological function it is an important difference between the acid-base properties and those associated with coagulation that whereas the former are common to many substances, the latter appear to be unique, peculiar to proteins. If it could be shown that protein coagulation is reversible, its biological significance would be greatly enhanced. Description of Coagulation. The coagulation of a protein occurs in two distinct steps:' first, a chemical change in the protein and second, precipitation of the altered protein. In the first step, the protein is so modified(whether by heat, acid, alkali, alcohol, or a number of other agents) that it is no longer soluble under conditions under which the original protein is soluble. Whereas the original protein is soluble a t its isoelectric point, modified protein is insoluble a t the isoelectric point. Addition of acid or alkali dissolves modified protein. The process whereby the original, or native, protein is changed is called denaturation and the changed protein is called a denatured protein. The distinctness of the two steps of coagulation is apparent when denaturation occurs at a hydrogen ion concentration removed from the protein's isoelectric point, for under these conditions even after prolonged heating, no visible change may be noticed. Denaturation has, however, taken place, and the 1
Chick and Martin: J . Physiol., 40, 404 (1910);43,
I
(1911).
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denatured protein has remained in solution, as can be shown by adjusting the hydrogen ion concentration to the isoelectric point, whereupon denatured protein precipitates. Although the second step in coagulation, precipitation of denatured protein, is readily reversible, the first step, denaturation, has been generally supposed to be irreversible. Flocculation of denatured protein depends primarily upon the change on the protein particles and is therefore probably analogous to the coagulation of colloids in general. Denaturation, on the other hand, is a process peculiar to proteins and should not be confused with the ordinary coagulation of colloids. Investigation of the kinetics of denaturation1 shows it to be a process following the course of a unimolecular reaction, the velocity of which is increased about 600 times when the temperature is raised 10%. Denaturation may therefore be slow at one temperature and rapid at a temperature only a few degrees higher. This is the reason why proteins appear to have a definite temperature of coagulation. Perhaps this remarkable temperature coefficient of denaturation will arrest the attention of chemists who may know of some analogous reaction; a t present it appears to be unique. It has accordingly been used to detect the r81e of protein in certain processes. When, for instance, bacteria are killed by heat, the process has a temperature coefficient of about 600 for I o O C , indicating that heat causes death in bacteria by denaturing certain bacterial proteins.2 The inactivation of some enzymes by heat has a temperature coefficient of 600 for IOT, indicating that denaturing a protein destroys enzymatic activity and that protein is probably part of the enzyme molecule. For crystalline pepsin this has been more directly demonstrated, for its loss of activity is quantitatively paralleled by its denaturation.3
A chemist will a t once ask what structural changes occur in denaturation. The process has been followed mainly by physico-chemical observations, particularly by the gross change in solubility, and little is known of the underlying structural changes. I n addition to the change in solubility it has been observed that denaturation causes a large increase in viscosity, a slight change, if any, in acid-base properties,i and, apparently, the loss of ability t o crystallize. In the case of hemoglobin the changes are especially conspicuous, for denaturation of globin, indirectly affecting the heme to which it is attached, causes a marked change in color and absorption spectrum and loss of the ability of heme to combine loosely with oxygen. It is not known what modifications in the chemical groups of globin bring about these changes. The only definite structural changes known to accompany denaturation are in the sulfhydryl group^.^ The presence of sulfhydryl groups can be demonstrated in denatured egg albumin by means of color reactions and oxidationreduction reactions. When native egg albumin is examined by the same methods these sulfhydryl groups cannot be detected. The same, or a similar Chick: J. Hygiene, 10, 237 (1910). Northrop, J. Gen. Physiol. 13, 739(1930). Booth: Biochem. J.,24, 158 (1930). 8 Arnold: Z. physiol. Chem., 70, 300 (1910).
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difference, can be demonstrated between the native and denatured forms of other proteins. We are now making a quantitative study of protein sulfhydryl groups. T h e Supposed Irreversibility of Coagulation. Ever since coagulation has been studied a t all, it has been taken as almost axiomatic that coagulation is irreversible, Usually coagulation has been thought to be the first step in the degradation, the breaking down of the protein. As a result, in the study of the chemistry of the normal, native protein coagulation has been considered something to be avoided. And in discussions of the physiological r61e of the proteins, coagulation has hardly been mentioned. I t is true that procedures which result in the denaturation of proteins also result in their degradation. Heat, which denatures proteins, also splits off ammonia. Acid which denatures proteins also splits off amino acids. Were such changes an inherent part of denaturation, then obviously reversal of denaturation would be impossible. But, as the Sorensens6showed, the splitting off of ammonia by heat has nothing to do with denaturation. It is a secondary change in the protein which takes place slowly when the protein is heated after it has already been denatured. I n general, there is no evidence in any case that the decomposition caused by denaturation procedures is anything but a secondary reaction which is entirely separate from denaturation itself. Reversal of denaturation cannot, therefore, be excluded as impossible on the a priori ground that denaturation involves decomposition. The only evidence that denaturation is irreversible has been that denaturation could not be reversed. It has long been familiar, for instance, that if an acid or alkaline solution of denatured egg albumin is brought to the isoelectric point, of egg albumin, all the protein precipitates. Indeed, whatever one does to denatured egg albumin apparently one cannot prepare native egg albumin from it again. This negative result might mean that denaturation cannot, be reversed. But it might also mean merely that egg albumin is unsuitable material for the experiment, that denaturation can be reversed only with difficulty, or that a special technique is required. The Reversal of the Coagulation of Hemoglobin. Our experiments' on the reversibility of coagulation had their origin in a simple but inconclusive experiment with hemoglobin. An alkaline solution of denatured hemoglobin was neutralized. Practically all the protein precipitated, but (as is not the case with egg albumin) a small fraction of the protein remained in solution. Consequently, the supernatant solution was slightly colored whereas when a precipitate of denatured hemoglobin is shaken with water, the water remains colorless. Furthermore, the spectrum of native hemoglobin is entirely different from that of denatured hemoglobin and the small amount of protein in the supernatant solution had unmistakably the spectrum of native hemoglobin. Either this small amount of hemoglobin had escaped denaturation in the first place or the coagulat'ion of hemoglobin had been partially reversed. Sorensen and Sorensen: C. r. Trav. Lab. Carlsberg, 15, No. 9, I (1925). 'Anson and Mirsky: J. Physiol., 60, 50 (1925);J. Gen. Physiol., 9,169 (1925);12, 2 j 3 1928;13, 121, 133,469, 477 (1930);Physiol. Rev., 10,506 (1930). 6
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The explanation of the result on the basis of incomplete coagulation became unlikely when, with improvement in technique, it became possible to convert not a few per cent but two-thirds and more of the coagulated hemoglobin into a protein with the characteristics of native hemoglobin. Such “reversed” hemoglobin is soluble, coagulable, and crystallizable. I t has the spectrum of native hemoglobin and can combine loosely with oxygen. The technique of the experiments is simple. I t consists in reversing the denaturation procedure by neutralizing an acid solution in two steps. Hemoglobin is readily denatured by acid. If an acid solution is rapidly and completely neutralized, that is, brought to the isoelectric point of the protein, then practically all the insoluble, denatured protein is precipitated. In fact, denatured hemoglobin is insoluble not only at its isoelectric point but in a wide region around its isoelectric point. If, however, the acid solution is not completely neutralized in one step, but is brought to the edge of the precipitation zone by adding just insufficient alkali to cause precipitation, then if, after a time, more alkali is added to complete the neutralization, only about a third of the protein is precipitated. The rest seems to be native hemoglobin. Similarly, if just enough alkali is added to make the solution just alkaline enough to prevent precipitation, then if, after a time, the solution is neutralized by the addition of acid, only a fraction of protein is precipitated. All these reactions can be followed with peculiar ease in the case of hemoglobin because hemoglobin is a pigment. The color of the solution tells immediately about how much protein it contains. The spectrum of the solution tells whether the protein is native or denatured. Hemoglobin consists of a colorless protein, globin, joined to an iron pyrrol pigment, heme. In the blood, hemoglobin transports molecular oxygen from the lungs to the tissues. Heme itself could not act as a carrier of oxygen. I t is not soluble enough and it does not combine loosely with oxygen in the way hemoglobin does. I t is by combination with globin that heme is modified to give hemoglobin its valuable biological properties. The globin must be native. The compound of heme with denatured globin (called globin hemochromogen and possessing its own type of spectrum) is not soluble in water and does not combine loosely with oxygen. Changes, then, such as denaturation of globin to which heme is attached in hemoglobin, are reflected in changes of the properties of heme, particularly in the spectroscopic properties which are readily and accurately followed. Heme in hemoglobin may therefore be used as a convenient and sensitive indicator of what is happening to globin. Just as globin influences heme with which it is combined, however, heme also influences globin, for instance by influencing the ease of its denaturation and the reversal of this denaturation. A priori it might even be true that the experiments on the coagulation of hemoglobin which have been described are concerned not with the general properties of the coagulable proteins but with the peculiarities of hemoglobin, peculiarities due t o the effects of heme on globin. All the available experimental evidence, however, indicates that the results obtained from the study of the coagulation of hemoglobin are entirely general.
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I n the first place, in all the ways which have been tried hemoglobin behaves like a typical coagulable protein. A11 the procedures such as heating, exposure to ultra-violet light and to high pressure, shaking, adding acid, alkali, alcohol, acetone, urea or thiocyanate, which convert egg albumin into an insoluble denatured protein have the same effect on hemoglobin. The heat coagulation of hemoglobin like that of egg albumin obeys the equation of a unimolecular reaction and has the extraordinary temperature coefficient of over 600. And denatured hemoglobin has the same general properties as denatured egg albumin. The Reversal of the Coagulatzon of other Protezns. The most convincing evidence that the hemoglobin reactions are not due simply to the presence of heme is that precisely the same experiments can be carried out even in the absence of heme. Colorless globin itself behaves just like hemoglobin. If an acid solution of denatured globin is rapidly and completely neutralized in one step, then practically all the protein is precipitated, especially if there is salt in the solution. If the two-step procedure is used, however, then a large yield of soluble, coagulable protein is obtained. With serum albumin the experiments are even easier. Denatured serum albumin is soluble except exactly at its isoelectric point or in the presence of concentrated ammonium sulfate. In neutralizing a solution of denatured serum albumin unless one has been very careful to secure the correct hydrogen ion concentration one does not observe any precipitate at all. T o obtain from denatured serum albumin a large yield of albumin which is soluble even in concentrated salt solution it is not necessary to neutralize in two steps. Simple, complete neutralization in one step suffices. One need not even let the solution stand before making the test for native protein. I n fact, it is hard in neutralization experiments with denatured serum albumin to avoid the reversal of coagulation. So long ago as I 910, Michaelis and Rona8 probably observed reversal. Unfortunately, there was some confusion at the time about two different kinds of coagulation. And the test for denatured serum albumin was uncertain. As a result, these experiments never received the attention they deserved. Later (at the same time as our hemoglobin experiments) Spiegel-kd~lf~gave good physicochemical evidence of the reversibility of the coagulation of serum albumin. More recently (unpublished experiments) we have studied serum albumin in a somewhat different way and have succeeded in obtaining from coagulated semm albumin, crystals of soluble coagulable protein in any desired amount. With egg albumin, no great amount of reversal has resulted from any procedure so far tried, a result in harmony with previous experience. Just as the great ease of reversal in the case of serum albumin seems to be associated with the great solubility of denatured serum albumin, so in the case of egg albumin the difficulty of reversal seems to be associated with the great insolubility of denatured egg albumin. Denatured egg albumin not only is insoluble over a wide range of hydrogen ion concentration around its isoelectric 9
Biochem. Z., 29, 494 (1910). Biochem. Z.,170, 126 (1926).
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point, but even beyond the zone of visible precipitation, it has a great tendency to form invisible aggregates (unpublished experiments). This is readily shown by viscosity measurements. In fact, the conditions for reversal in the case of other proteins are, in the case of egg albumin, the conditions for gel formation. It is possible t o obtain a clear gel from a solution of denatured egg albumin containing only a half a per cent of protein. The ease of reversal then, varies from one protein to another; from one hemoglobin derivative to another; from the hemoglobin of one species to that of another; from serum albumin to egg albumin. Such differences in the ease of reversal are not surprising since, as is well known, there are similar differences in the ease of denaturation. Khere reversal is possible, it is brought about by keeping the protein for a time in a solution that is beyond the precipitation or aggregation zone and yet not too far from the isoelectric point. With denatured egg albumin it does not seem possible to realize such conditions. It remains to describe the more detailed evidence in favor of the reversibility of coagulation. Clearly it is not sufficient to obtain from a protein which seems to be denatured a protein which seems to be native. There must be adequate tests for the completeness of the coagulation and for the identity of the protein obtained from the coagulated protein with the original native prot'ein. To obtain completely conclusive results is a t present impossible. S o t enough is known either about the changes involved in denaturation or about the means of characterizing completely a native protein. Evidence of the Completeness of Coagulation. Since the most conspicuous change associated with denaturation is the change in solubility, the most obvious test for complete denaturation is insolubility. This test has been made in all cases. Csually an acid solution was rapidly neutralized with resulting complete precipitation of the protein. I n one experiment, it was possible to test for insolubility under exactly the conditions under which reversal took place. To an acid solution of denatured hemoglobin there was added an excess of alkali just sufficient to keep the protein in solution. If this solution was half saturated with ammonium sulfate immediately, practically all the protein was precipitated. The later the salt was added, the less protein was precipitated. Finally, two-thirds of the protein remained in solution. I n other words, denatured hemoglobin originally brought into slightly alkaline solution was insoluble in half saturated ammonium sulfate. With time it gradually changed into another form which like native hemoglobin was soluble. It might be objected that precipitation at the isoelectric point is not an adequate test for insolubility or denaturation. Conceivably, when a denatured protein is precipitated it might carry down any native protein present. To explain reversal on this basis one must assume that only one-third the protein is denatured and that this third, when precipitated carries with it two-thirds which escapes denaturation. Experimentally, however, there is as yet no evidence that this can take place. If one prepares a known mixture of native and denatured hemoglobin or globin and precipitates the denatured protein, the concentration of native protein in the solution is not changed.
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About the same yield of soluble protein is obtained from denatured protein if it is left in acid three minutes or eighteen hours; if it is heated, or left in acid, or heated in acid at two different temperatures; if it is denatured by urea, or trichloracetic acid, or 90 per cent acetone containing acid. If obtaining soluble protein were simply an indication of incomplete denaturation the yields ought to vary enormously with the time to which the protein is subjected to the denaturation procedure and with the nature of the denaturation procedure. The only alternative conclusion is that complete denaturation is impossible with the ordinary procedures. I n the case of denaturation by urea, it is possible to follow the course of denaturation by viscosity measurements. Concentrated urea solutions denature proteins slowly and also keep denatured proteins in solution, even a t the isoelectric point. As the protein remains in the urea solution, more and more of it is converted into a form which is insoluble in the absence of urea. Associated with this formation of insoluble, denatured protein there is a gradual increase in the viscosity of the urea solution. Finally, all the protein is precipitated if the urea is removed. From this time on there is no further change in the viscosity. The hemoglobin precipitate formed by removing the urea after the viscosity has ceased to change may be largely reconverted by the reversal procedure into soluble, coagulable protein. Lastly, we have recently been testing the completeness of denaturation by measuring the extent of specific group changes. Evidence of the Completeness of Reversal. I n all, then, the evidence so far obtained indicates that the experiments on the reversibility of coagulation have been started with completely coagulated protein, that a t least no significant part of the soluble protein finally obtained is derived from protein which never was denatured. To complete the proof of the reversibility of coagulation it must be shown that the soluble protein finally obtained is the same as the native protein originally coagulated. I t might be true that some soluble fraction was extracted from the coagulated protein, or that some parts of coagulation were reversed--enough to give a soluble protein-but that the “reversed” protein was still quite different quantitatively from the original protein. Normal hemoglobin and “reversed” hemoglobin have accordingly been compared in quite a number of ways, some of them possible only because of the presence of heme in the molecule as an indicator. I n the first place “reversed” hemoglobin is not only soluble, but it can by heating be coagulated again. The temperature of coagulation is exactly the same as that of normal hemoglobin. Similarly “reversed” hemoglobin can be crystallized (no denatured protein has yet been crystallized) and the crystals to the unaided eye seem to have the same form as those of normal hemoglobin. Precise crystallographic measurements have not been made. The spectrum of “reversed” hemoglobin has the same pattern as that of normal hemoglobin. The bands are in the same positions within the small experimental error of two k g s t r o m units. Furthermore, “reversed” hemoglobin can be converted into pigments having the same characteristic spectra as the derivatives of normal hemo-
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globin. “Reversed” hemoglobin, like normal hemoglobin but unlike denatured hemoglobin] can combine loosely with oxygen. It has less affinity for oxygen than for carbon monoxide and the ratio of the two affinities is the same as in the case of normal hemoglobin. The tests which have just been described are sensitive enough to distinguish readily between the slightly different hemoglobins of different mammals. Yet they cannot distinguish between “reversed” and normal hemoglobin. It is interesting to notice that although the spectra of the different native hemoglobins are measurably different, the spectra of the different denatured hemoglobins are not measurably different. This is in harmony with immunological experiments which show a decrease in species specificity on denaturation. When the denaturation of the different hemoglobins is reversed] the original spectra and hence the original spectral specificities reappear. The comparison of “reversed” globin with normal globin cannot be undertaken a t present because there is not yet available any certainly normal globin. The separation of globin and heme by present procedures seems to involve the denaturation of the protein. Hill and Holden’O believed that they obtained native globin directly from hemoglobin. But no evidence was given in support of this claim. One can, however, synthesize hemoglobin from “reversed” globin and heme and compare the synthetic with normal hemoglobin. We have crystallized synthetic methemoglobin and carbon monoxide hemoglobin but the comparison with normal hemoglobin has not yet been completed. The nativr globin of hemoglobin obviously has specific chemical groups with which it combines with heme to form hemoglobin. Denatured globin does not have these groups. At any rate, it cannot combine with heme to form hemoglobin, a substance with the characteristic spectrum and other properties of native hemoglobin. “Reversed” globin can once more form hemoglobin. There is reversal] then, as tested by examination not only of the physical but also of the specific chemical properties of the protein. Recently, we have also been measuring the thiol groups of native and denatured globin and serum albumin to see whether there is reversal of the characteristic changes in these groups. So far, then, no differencehas been found between normal and “reversed” protein. I t is, of course, possible that differences might be found were the tests which have been used made more accurate or were other tests used. There is always the difficulty that since it is not known precisely what changes take place in denaturation, it is not known what differences might conceivably exist between normal and “reversed” protei?. The most general sort of test which we have been applying recently is the solubility test. If native and “reversed” proteins are different] regardless of the nature of the difference] then their solubilities should be different. ~~
~~
Biochem. J., 21, 625 (1927).
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We may summarize the experiments on the reversal of coagulation in the following manner: If an acid solution of denatured egg albumin is neutralized] all the protein is precipitated, the denaturation of egg albumin being apparently irreversible just as has always been supposed; but if hemoglobin, globin or serum albumin is denatured by the ordinary procedures, one can, by a suitable technique, obtain from these insoluble denatured proteins, soluble, coagulable proteins which seem to be the same as the original native proteins. Experiments not yet completed or others not yet attempted may change the present conception of denaturation or disclose differences between native and “reversed” proteins. So far as the present evidence permits a conclusion, however, coagulation seems t o be reversible. The only reason there ever was for believing coagulation to be irreversible] namely that coagulation could not be reversed, no longer has an adequate experimental basis. If the theory of the irreversibility of coagulation is still to be supported, it must be on the basis of new experiments] experiments which explain the results obtained with hemoglobin] globin and serum albumin. The Rzological Significance of Coagulation. It is a familiar fact that the solid matter of living active tissue consists mainly of coagulable proteins. Denaturation produces gross changes in the properties of proteins. Denaturation seems to be reversible. With these considerations in mind, it is difficult to believe that Nature has not exploited the coagulability of proteins. I t is, for the moment, a tempting hypothesis that denaturation and its reversal are biological reactions which are important in ordinary cellular processes. It is thjs hypothesis a t any rate which has led us to the study of the tissue proteins from the standpoint of coagulation. One wants to know whether the coagulation of the tissue proteins can be reversed, what group changes accompany this coagulation, how these changes can be studied under physiological conditions] and what is the nature of their function. The Laboratories of The Rockefeller Institutejor Medical Research, Princeton, N . J . and The Hospital of the Rockefeller Institute for Medical Research, A’ew York. N . Y .
