The Denaturation of Albumin

T H E DENATLRATIOK O F ALBCiLlIK* BY WILDER D. BANCROFT AND J. E. RUTZLER, JR.

1. Denaturation by Heat Man is incurably empirical. When a given experiment fails to produce the expected result, he says it will not work. Whereas, were he neither empirical nor dogmatic he would say that it does not work. When egg albumin or egg white, as the case may be, is heated to a certain temperature, coagulation of the suspended particles takes place. Because people, in the past, failed to repeptize the gel so formed, it has been tacitly assumed that it cannot be restored chemically unaltered, a t least, to the original colloidal state. Some profound change has been held responsible for this failure of the albumin to repeptize. This apparent set of circumstances left an opportunity open to coin a word; the term “denaturation” or “denaturization” resulted inevitably. Much has been written about “denaturation”, for instance, the fol1owing:l “One of the most characteristic properties of the albumins is the physical instability of their solutions and their marked tendency to revert to a solid or semi-solid state. This change may be effected by apparently trivial factors, such as evaporation, contact with porous substances, etc. In this respect also the albumins behave very much like the inorganic colloids. This change (irreversible coagulation) can be brought about a t once by the application of heat, and it is important to note that all true natzae albumins can be coagulated in this manner. After coagulation their solution can only be effected by influences which lead t o their more or less extensive destruction. They have lost those physical properties which characterized them individually as albumins; they are permanently denaturzzed, as Neumeister expresses it.” On page 3 2 , Simon continues: “Coagulation is not an essential phase of denaturieation. Denaturization may indeed become manifest by the nonoccurrence of coagulation. Denaturized and coagulated albumins can only be brought into solution by means which will at the same time produce integral changes in their composition, via : by means of proteolytic ferments, dilute mineral acids or alkalies, concentrated organic acids under the application of heat, etc. “The nature of the process which determines denaturization is possibly a primary cleavage. Nobhing certain, however, is known.” It would seem from this quotation that denaturation is a process occult and more or less unintelligible.

* This work is part of the rogramme now being carried out at Cornell University under a grant from the Heckacger Foundation for the Advancement of Research established by August Heckscher at Cornell University. 1 Simon: “Physiological Chemistry,” 30 (1904).

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Writing of “Denaturation and Coagulation”, Miss Lloyd’ says : “Coagulation is a physical condition, and by suitable means coagula can be redispersed or dissolved. The solution of coagulated protein, however, differs very definitely from that of the original material from which it came. Coagulation is not in itself an irreversible process, but in all cases is preceded by an m e verszble chemical change zn the protezn known as denaturataon.2 “Under the heading of ‘denaturation’ can be included a number of reactions, the common features of which are a complete loss of solubility in water and in dilute salt solutions. Denatured proteins, however, are readily dissolved in dilute acids or alkalis, giving viscous colloidal solutions which react towards electrolytes as if they were of the suspensoid type rather than the emulsoid type characteristic of normal proteins. The change involved in denaturation seems to be a structural alteration in the protein molecule, which leads to a re-arrangement of the linkages in the molecule, but not an actual degradation. This change is accompanied by a complete loss of the power of swelling by the imbibition of water. . . . Proteins can be denatured by strong acids or alkalies, by salts of the heavy metals, by heat, by light, by mechanical agitation, by pressure or adsorption on a surface, and by the action of alcohol or acetone. The interrelation between qifferent types of denatured proteins has not yet been worked out.’ ” On page 2 2 9 Miss Lloyd continues:“The commonebu example of this [heat denaturation] is, of course, the setting of the white of an egg which takes place on boiling. Denaturation and coagulation are both influenced by temperature, time, the reaction of the solution, the presence of water and by the nature and concentration of the electrolytes present; but the effect of these factors is different for each of the two processes.” Bechhold3 also makes the point that when proteins are denatured they lose their capacity to swell. On page 163, Bechhold makes the definite statement that, “reversible coagulation is purely a salting out, whereas one must assume a chemical change with irreversible coagulation.” Young4 maintains that denaturation is a primary chemical change. Anson and Mirskys make the statement that, “denaturation is not a general disintegration of the native protein.” In a second paper the authorsB come to the conclusion that denaturation and its reversal are very obscure chemical changes. We find Robertson7 on record as follows: l ‘ . . . which leads to the apparently irreversible character of the process (dehydrating proteins by heat) ; apparently and not actually irreversible because, as Corin and Ansiauxs have “Chemistry of the Proteins,” 2 2 8 (1926). Hardy: J. Physiol., 24, 158 (1899). Bechhold: “Die Kolloide in Biologie und Medirin,” 161 (1929). Proc. koy. Soc., 93B, 235 (1922). J. Gen. Phykiol., 13 11, 121 (1929). J. Gen. Physiol., 13 11, 133 (1929). ”‘The Physical Chemistry of the Proteins,” 308, 309 (1918). *Bull. Acad. roy. Belq., (3) 21, 321, 345 (1891).

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WILDER D. BAKCROFT AND J. E. RUTZLER, JR.

shown, if a solution of protein be cooled and vigorously shaken just as the first traces of heat coagulation appear, the incipient coagula will again pass into solution. The apparent irreversibility of the later stages of heat-coagulation is probably attributable to the high internal friction of the floccula which are formed, leading to extremely slow molecular movement and the introduction of a time element of very considerable magnitude.” Cohnheim’ says that albumin cannot be put back into solution without extensive changes and splitting; it is permanently denatured. The denaturation of serum albumin solution by heat can be retarded by adding sucrose or glycerol to the system.* The physico-chemical properties of the native proteins are left more or less unchanged as a result. The protective action of the glycerol was found to be less than that of the sucrose. Further, with sufficient concentration of sucrose, rabbit serum proteins were found to be completely thermostable at 6 2 ’ ; while egg albumin, under like conditions, was thermo-stable at 75’. Albumin is prevented from coagulating3 even in boiling water by the presence of starch. Coagulated egg albumin was found to redissolve when heated to 148’ with a small quantity of water. Chick,4 working with pseudo-globulin, derived from horse serum, makes the statement that denaturation cannot be identified with any chemical changes in the material; whereas we find that \Vu5 concludes that the essential step in denaturation is a hydrolytic fission of the protein, and the essential step in coagulation is molecular condensation. Earlier Chick and Martin6 maintained that the fact that the hydrogen ion concentration diminishes after heat coagulation points to the view that denaturation is a chemical change. “It, is not a t present easy to form a satisfactory theory of denaturation,” are the words Miss Lloyd? uses in launching a discussion of the theories of denaturation. The author then mentions three prevalent theories, the opening up of internal anhydride rings, the closing of rings with the consequent formation of internal anhydrides, and hydrolytic cleavage, none of which are adequate to explain all the facts. From this short review it becomes obvious that the behavior of albumin, as a result of which it supposedly becomes either irreversibly coagulated or chemically changed, is not, clearly understood. People are at variance as to the mechanism; and cause and effect are sometimes apparently confused. The whole problem has been approached from the viewpoint of true solutions, or colloidal ions, or amphoteric electrolytes. The problem has not been considered logically from a strictly colloid chemical viewpoint. “Chemie der Eiweisskorper,” (191I ) .

* Beilinsson: Biochem. Z., 213, 399 (1929). Kingzett: “Animal Chemistry,” 380 (1878). Biochem. J.,8, 404 (1914). H. Wu: Chinese J. Physiol., 3, I (1929); Chem. Abe., 23, 4952 (1929) J. Physiol., 40, 404 (1910). “‘Chemistry of the Proteins,” 243 (1926).

THE DENATURATION OF ALBL-MIN

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Rancroft,’ in discussing the effect of electrolytes on albumin, says: “In other words, we will give up, for the time being, the dogma that albumin is an amphoteric electrolyte.” Further, “the precipitation of a colloid is always reversible in case no coalescence or agglomeration takes place, because one of the standard methods of preparing a colloidal solution is to wash out the excess of the precipitating agent. Now the more strongly the precipitating agent is adsorbed, the harder it is to wash it out and consequently the more nearly irreversible the precipitation is.” Electrolyte-free albumin is said to be very stable toward the action of heat,* although PauliS disputes this finding. Also, it is well known, that electrolyte-free albumin does not coagulate on standing. Concerning this Bancroft says: “The only way that I see to account for the stability of the electrolyte-free albumin is to postulate that in the absence of salts the peptizing action of water is enough to keep the albumin in colloidal solution. “It does seem to me, however, that it will be an easier task to account for the properties of electrolyte-free albumin on the basis of varying adsorption than on the basis of an amphoteric electrolyte”. Wo. Ostwald‘ takes a step in the right direction when he says: “Albumin sols are usually amphoteric, that is, they must be either positively or negatively charged, depending upon certain conditions,” although the way the word amphoteric is used is somewhat unfortunate. Kruytj is quite positive in his discussion of proteins as colloids. “There is, however, a special reason which has led many investigators to look upon emulsoids as molecularly dispersed systems, more particularly, as electrolyte solutions; this is the fact that a great many properties of protein solutions may be explained by considering these solutions as systems of amphoteric electrolytes. It may be pointed out at this place that a discussion of emulsoids in terms of ionically dispersed systems must necessarily conflict with typically colloidal properties of these systems. “The main criticism that can be raised against the concept of the electrolyte nature of protein solutions lies in the fact that the division of colloids into suspensoids and emulsoids would then be without continuity. In other words, we should have to ascribe to colloid chemistry a dualistic line of reasoning which is highly unsatisfactory. Whereas, for instance, the electrical properties of suspensoids are to be explained by means of the electroadsorptive phenomena of the peptizing ions which divide themselves between the particles and the medium . . ., we should have to consider for protein solutions an ionic equilibrium of one single kind of molecules dissociated into ions, the degree of dissociation being governed by Ostwald’s dilution law.” Denaturation, in review, is defined as the irreversible precipitation of a protein sol. Likewise, coagulation is defined as the reversible precipitation I

J. Phys. Chem., 19, 349 (1915). Schmidt: Pflugers Archiv, 8, 75 (1874); Heynsius: 9, 514. Beitr. chem. Physiol., 10, 53 (1907). “Practical Colloid Chemistry,” 151 (1924). “Colloids,” 178, 179 (1927).

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of a protein sol. There is an experiment which apparently illustrates this difference between denaturation and coagulation. Pauli and Handovsky’ boiled an egg albumin system which was 2-normal in respect to potassium thiocyanate. There was no coagulation. A control system and a portion of the treated sol were dialyzed against running water. The control system remained clear; whilst, unfortunately, there was extensive flocculation in the treated sol. The boiled, undialyzed system containing potassium thiocyanate is considered to be denatured but not coagulated. Denatured serum albumin has been reversed by Spiegel-hdolf.* The serum albumin was treated with dilute sodium hydroxide solution. “The experiments were carried out thus: Varying quantities of the same heat denatured albumin were dissolved in I O O cc. of 0.01N XaOH in exactly the same way. I t then appeared that the absolute content of the protein of the resulting solutions decreased with the decreasing quantity of the proteins employed, while a constantly decreasing residue of protein remained undissolved. If these liquids were electrodialyzed, t’he quantity of protein remaining in solution was found to decrease with a decrease in the initial quantity of heat-denatured protein used, and finally to disappear. That is, from the solution of 0.5 gram of heat-denatured albumin in IOO cc. of 0.01N NaOH, t’here results a protein solution which is quantitatively precipitated on electrodialysis. There exist, therefore, two limiting cases, within which there are continuous transitions. With a sufficiently large excess of protein, we get only water-soluble protein; with a correspondingly large excess of alkali, only a water-insoluble protein.” The water soluble protein was obtained by dissolving 4 grams of serum albumin in I O O cc. af 0.01IS NaOH. “From this physico-chemical evidence, the product in question (the watersoluble protein) is not distinguishable from true albumin. “In order, however, to try a further criterion for the characterization of protein X, the highly sensitive immuno-biological methods were employed. The precipitin method was select,ed.” The experimental results show that both horse serum-albumin and the electrodialyzed sample (4 grams of protein dissolved in I O O cc. of 0.01 X NaOH) behave as antigens toward the precipitin up to a dilution of one part of protein to one hundred thousand parts of physiological salt solution. The negative result of the control test demonstrates the specificity of the reaction.” The author then summarizes, p. 3 I I : “Accordingly no essential difference is demonstrable between protein X and its parent material (a true serum albumin) by the physico-chemical and immuno-biological methods here employed. The agreement is so close that, provided no new experimental results arise to contradict it, we are forced to admit an actual identity of the two proteins. Such an identification, however, would imply that the changes produced by heat had been made chemically reversible by the treatment given the heat-coagulated albumin.” Bechhold: “Die Kolloide in Biologie und Medizin,” 170 (1929).

* Alexander: “Colloid Chemistry,” 2, 309 (1928).

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I n an attempt to explain the chemical changes which have supposedly taken place, the author says on page 313: “The demonstration that the changes brought about by heat can be reversed by small amounts of alkali or acid, supports the view that ring closure accompanies the heat-change, For, while the action of these added substances, which persists even after their electrodialytic removal, can be interpreted as an hydrolysis, there might possibly have been involved a breaking down of a pre-existing ring structure, On the other hand, it is not obvious how any previously occurring hydrolysis can be completely reversed by an added substance which itself is known to aid hydrolysis. The above findings seem to strengthen the theory that the change appearing upon heat-denaturation of the proteins depends upon a ring closure of the groups involved.” The reversal of denaturation is obviously a paradox. It can be accounted for in two ways. I n the first place, the experimental results of SpiegelXdolf can be discounted. This seems to be highly improbable. Refusing to challenge these results brings up the question as to whether the term denaturation is really accurate or not. This will be taken up later. Wilhelm’ predicted, in 192 j , that it should be possible, under the correct conditions, to reverse denaturation. In a later paper2 he made good on his prediction and reversed heat-denatured serum albumin. The coagulated albumin was prepared by electrodialysis in a hot water bath, or over a free flame. The gel was repeptized by heating it in boiling water containing a definite amount of salt. The salts used were potassium thiocyanate, sodium salicylate, and sodium benzoate. The systems are defined, for example, as follows: i . 2 mg. of albumin, dissolved in enough z . r j X K C S S solution so that the K C S S content was 1.06 grams; the same amount of albumin was dissolved in enough normal sodium salicylate solution so that the sodium salicylate content was 0.32 gram. In order to obtain a clear solution of the coagulated serum albumin these concentrations (in addition to several other concentrations which the author tabulates) had to be rigidly adhered to; greater or lesser amounts would not accomplish the results. After dialysis of these systems containing peptized albumin, the albumin was found to be again coagulable by heat. It’ was found that the same salts which peptize the coagulated albumin also prevent its coagulation, much less salt being necessary for the latter than for the former. I t is a noteworthy fact that Wilhelm found that both the total quantity and the concentration of peptizing agent with respect to the albumin must be fixed, as is shown from the data above. From the results obtained, it was concluded that the peptization of the coagulated protein is influenced by two distinct processes. There is first an imbibition of water by the protein; the swollen protein then adsorbs ions from the solution becoming peptized thereby. Biochem. Z., 180, 231 (1927). Kolloid-Z., 42, 217 (1929).

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The results of U‘ilhelm controvert the conclusion to which Pauli’ came concerning the two processes, denaturation and coagulation. Denatured hemoglobin was repeptized by Anson and Mirsky.2 Horse hemoglobin was used in this work. Either sodium hyposulphite or potassium cyanide was found to be apparently indispensable to the repeptization of the denatured protein. The system was extracted with toluene during the process. Carbonyl hemoglobin was finally crystallized from repeptized systems which were denatured by heat, acid, and urea. The reversed protein was shown to be identical with the original : by crystallizing the carbonyl derivative, by its restored heat coagulability, and by the fact that it denatured again with acids and alkali. The conclusion is reached that the coagulation of proteins, in general, is a reversible chemical change which is very obscure. In another paper Anson and Mirsky3 discuss denaturation a t greater length. They state that it has not yet been proved that the various forms of denaturation are due to the same changes in the protein, for concentrated solutions of urea denature proteins but keep the denatured proteins in solution. When the urea is dialyzed out, the protein precipitates completely. Anson and Mirsky say that the denaturation of hemoglobin is exactly the same process as the denaturation of albumin. They also say that KI, KCSS, and sodium salicylate probably denature proteins in general, when the protein solution is buffered at [H+] = I x IO^.', or near the isoelectric point. Apparently urea can denature egg albumin, and likewise dissolve the denatured albumin. Egg albumin was allowed to stand in a concentrated urea solution for a day. X few drops of this solution were added to 15 cc of water which was buffered at [H+] = I X IO-^.^. This caused a heavy precipitation. Upon dissolving some urea crystals in the water, t’he protein redissolved. In these two papers t,here is no mention of peptization, the view-point being strictly one of chemical reaction. The problem to repeptize heat-coagulated egg albumin not having been solved, the present work was undertaken with two purposes in view, i.e., to accomplish this repeptization, and to endeavor to show that so-called denaturation is a colloidal phenomenon and not due to a chemical reaction. For the purpose of t,his investigation the principles of colloid chemistry will be adhered to as rigidly as the subject will permit, and “the dogma that albumin is an amphoteric electrolyte” will be discarded. It has been shown that it is possible to repeptize serum albumin gels by the use of a variety of reagents and to obtain solutions which are identical with the original. Considered as a problem in colloid chemistry, there is no good reason why this should not be so. This theme will be dealt with more completely further on. 1

3

Hofmeister’s Beitrage, 9, 415 (1908). J. Gen. Physiol., 13, 11, 133 (1929). J. Gen. Physiol., 13, 11, 121 (1929).

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Experimental Study I n order to observe experimentally the manner in which heat-flocculated egg albumin and egg whit,e behave, from the colloidal standpoint, some rough qualitative experiments were undertaken first. A portion of the white of a hard-boiled egg was ground in a mortar t,o a fine powder with solid potassium iodide and cane sugar. Distilled water was added to the mixture. h cloudy sol formed which cleared up somewhat upon the addition of more sugar. Upon performing this experiment several times it became apparent that t’he amount of peptization depended, as one would expect, to a large extent upon the degree of fineness to which the system was reduced upon grinding. The selective wetting of heat-denatured, globulin-free albumin by several liquids was next determined. Glycerol and water both wet albumin. When dried and finely ground albumin is put in contact with water the particles show a tendency to form lumps, to agglomerate. With glycerol, however, there is apparently little or no agglomeration. Small samples of albumin were put in contact with chloroform, acetone, and benzene. Upon adding glycerol to each of the systems, the albumin immediately passed into the glycerol layer. Thus, glycerol wets egg albumin preferentially to these liquids. In view of this, the heat-coagulated albumin must be considered as still being hydrophylic, although Bechholdl states that heat coagulation changes natural proteins, which are hydrophilic, so that they become hydrophobic. What actually happens is that they lose most of their water sheath. Cold coagulation (Kaltefallung) of albumin is said to be irreversible. However, it was found that freezing the white of an egg and then lowering the temperature below minus IO’ did not visibly affect the physical condition of the sol when it again comes to room temperature. It will later be shown that this is exactly the behavior that would be expected of such a sol. Following the lead of Spiegel-Adolf,2 we tried to repeptize heat-coagulated egg albumin with sodium hydroxide solution. The globulins were precipitated out of an absolutely fresh egg-white sol by the approved method. The globulin-free albumin was coagulated by heating it in boiling water for fifteen minutes. The lumps of coagulated albumin were filtered off and washed several times with boiling distilled water. Five systems were prepared using this sample; 16 grams of the wet sample were put into a flask with IOO cc. of distilled water and 0.; gram of sodium hydroxide; I O grams of the wet sample were put with the same amounts of sodium hydroxide and water; five grams of the wet sample were put with one-half the amount of sodium hydroxide and the same amount of water; 0.5 gram of the albumin was put with 5 % of the amount of sodium hydroxide in the same amount of water, and finally; 16 grams of the wet albumin were put with 2.1 grams of sodium hydroxide and IOO cc. of water. All of the systems, except the last, were agitated on a shaking machine for 3 days. The last system became clear, with all of the “Die Kolloide in Biologie und Medizin,” 164 (1929). Alexander: “Colloid Chemistry,” 2, 309 8.(1928).

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albumin apparently peptized, in the course of 1 7 hours. The others failed to peptize to any marked extent. The system containing the peptized albumin was dialyzed against running distilled water for about 30 hours. The sol was then found to have the following physical and chemical characteristics. I t was neutral to litmus, alkaline to methyl orange, and slightly alkaline to phenolphthalein. I t was odorless, and absolutely clear and colorless. The xanthoproteic and biuret reactions were positive. I t coagulated with heat only upon making it acid to phenolphthalein but not adding enough acid to make it acid to methyl orange. Ammonium sulphate was used to change the alkalinity of the sol. The addition of glacial acetic acid caused a heavy precipitation. I n the presence of alcohol and sodium chloride, the sol did not coagulate when heated to above 78’. I t required less than half-saturation with ammonium sulphate to precipitate large quantities of the colloid. Because of the slight dissimilarities between this sol and uncoagulated albumin we cannot at the present time conclude that the albumin did not suffer some slight hydrolytic cleavage during repeptization. The statement’ has been made that ether denatures egg albumin. Upon investigating this phenomenon quite the reverse was found to be true, under certain conditions. Fresh egg white was diluted with distilled water to form a IO% sol. Globulins, of course, precipitated; they were filtered off as completely as possible. The sol was extracted twice with ether. Quite a stable emulsion persisted a t the ether-sol interface. The ether was gently boiled out of the extracted sol. The addition of alcohol to this sol produced no coagulation whatever. Alcohol did precipitate it when enough was added so that the dispersion medium was more than a 50% solution of alcohol. The effect of heat was determined by immersing the sol in a test tube in boiling water for 15 to 20 minutes. The sol remained clear; absolutely no coagulation took place. This was too good, so that the obvious explanation that most of the albumin had concentrated a t the interface was tested. The xanthoproteic reaction was tried on the sol. The addition of nitric acid resulted in heavy coagulation. To check this test, glacial acetic acid was added to the sol; again a large amount of precipitate came down. The whole experiment was repeated several times with the same results. h portion of the sol which was not extracted with ether coagulated normally upon immersing the test tube containing it in boiling water. This method of preventing coagulation was tried on a soyc egg white sol (50 cc. egg white diluted with 50 cc. of distilled water). The result was the formation, upon heating, of a slight amount of coagulum, which was manifest by the appearance of a heavy cloud in the sol. A far larger amount of precipitation occurred upon the addition of either acetic acid or nitric acid. A striking difference in properties between the native egg-white coagulum and that produced when an ether-extracted sol was coagulated by heat in the 1

Lepeschkin: Kolloid-Z., 32, 100 (1923); Hammarsten: “ A Textbook of Physiological

Chemiatry,” 107 (1914).


I53

presence of a little acetic acid is observable. The untreated sol coagulatks into lumps, or into one solid mass, depending upon the conditions. The ether-treated sol, when coagulated by heat in the presence of a little acetic acid comes down in the form of strings. The dense precipitate is apparently more highly hydrated than the string-like precipitate; its probable classification is as a curdy’ precipitate. The other precipitate would then be classified as a flocculent precipitate, or as will be shown in a later paper more probably as a jelly. In this case there is apparently a rather sharp line between flocculent and curdy precipitates. The ether under this classification must stand trial for the removal of something which makes the water sheath of the colloidal albumin particles less strongly adsorbed; or else the ether itself is held by the albumin thus making its water sheath less strongly adsorbed. Coagulation, under this last hypothesis, must start before the ether is removed from the albumin particles by t h e heat. I t has been shown that ether can be used to prevent heat coagulation. If heat coagulation were due to a chemical reaction, it is difficult-to see just how the ether extraction of the sol would prevent it. How ether could react with an amino group, an imino group, a carbonyl group, a sulphhydryl group, a carboxyl, or phosphorus group is not a t all obvious. Further, how it could, in this case, form a heat-stable addition product, cause hydrolysis, or open an internal anhydride ring is equally hard to see. Also, the ether was free from peroxide by actual test; so that that could not have been responsible for the condition which was brought about. Therefore, the burden of the proof is upon him who claims that this phenomenon is due to a chemical reaction. The ether removes something from the albumin sol. There was a distinctly measurable amount of residue upon evaporation of the ether which was used for the extraction. The solid material is apparently soluble in ether and peptizes in water; for the solution remained clear until most of the ether was evaporated off. Then, when the water content became greater than the ether content, the system became cloudy. The sol so formed was diluted with water and an electrophoresis experiment performed upon it. The sol was discharged from both electrodes with a greater amount of discharge from the anode than from the cathode. This must mean that there are two substances present, one charged negatively and the other positively, the one charged negatively predominating. The concentrated extract apparently coagulates a 107~ albumin sol, for it became decidedly more turbid upon addition of the extract. It is interesting to note the results obtained by an electrophoretic study of three lecithin sols. The lecithin was prepared from egg yolk.* The first sol was prepared by shaking lecithin with water and drawing the air out of it by means of a vacuum pump. As was e ~ p e c t e dthe , ~ suspended particles travelled toward the anode, showing that they were negatively charged. The second

’ Bancroft: “Applied Colloid Chemistry,” 193 (1926). 3

Webster and Koch: “Laboratory Manual of Physiological Chemistry,” I (1903). Kakiuchi: J. Biochem. (Japan), 1 , 165 (1922).

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sol was prepared by adding an alcoholic solution of lecithin to about a 2 0 7 ~ alcohol-water solution. The suspended particles were apparently discharged at both the anode and the cathode, with the larger amount of discharge at the anode. The third sol was the same as the first; only it was heated to about 90’ before the electrophoresis was started. I t behaved, not like the first sol, but like the second. An analysis of egg white‘ shows that the amount of fat, including lecithin, present is 0.25%. From these facts it is obvious that the thing partly responsible for heat coagulation may be a fatty material associated with lecithin, but having under certain conditions an opposite charge. The plus charge that this substance has would then be partly responsible for the flocculation of the protein from suspension upon heating. When one allows undiluted egg white to stand with enough ether for an appreciable time, it does, as has been maintained, coagulate. This is quite obviously due to the removal of water, not from the albumin molecule, but from the sheath of water surrounding the individual suspended albumin particles. The white of an egg was diluted j o % with distilled water and coagulated by immersion in boiling water. Part of the substance prepared in this manner was extracted for eight hours with peroxide-free ether, by merely allowing the ether to stand in contact with the egg white, and changing the ether frequently. The ether was drained off of the sample and a portion of it was put in 1 2 5 cc. of distilled water containing a fairly large amount of ammonium thiocyanate. The system was agitated on a shaking machine. In about 24 hours the appearance of the sol indicated that considerable peptization had resulted. The ether extraction of a portion of the coagulated egg white was continued until the ether, as a result of many changes, dried the egg white to a hard, transparent mass. Three experiments were performed upon this material. A small amount of it was put into each of the following: 1 5 cc. of water containing about two grams of ammonium thiocyanate, 1 5 cc of water containing about two cc. of ammonium phosphate solution (prepared by neutralizing syrupy phosphoric acid with ammonium hydroxide and adding enough base to produce a faint ammoniacal odor). The three test tubes were shaken on a machine for three and a half days. At the end of this time it could be seen that in the case of the latter two experiments peptization occurred, whereas, with the pure water there was no peptization. The three systems were heated to the boiling point. The system containing only albumin and distilled water became quite cloudy during this process; the other two did not change markedly. Another portion of the coagulated egg white described in the paragraph above was extracted with peroxide-free ether for three days in a Soxhlet extractor. A portion of the albumin thus obtained was placed in water and the system boiled for about ten minutes. A cloudy sol formed. It was further broken up by stirring by means of a high-speed soda mixer. This was Hutchinson: “Foods and Dietetics,” 148

(1900).


‘55

followed by shaking for 1 2 hours on a shaking machine. The resuIt was the formation of a very cloudy albumin sol. Heating separate portions of this sol with ammonium thiocyanate, potassium iodide, and starch revealed the fact that the sol became less cloudy (accompanied by no precipitation) in the case of the first compound but did not change in appearance with the other two. The sol containing ammonium thiocyanate was acid to litmus. Sodium bicarbonate was added to neutralize it. This resulted in the liberation of ammonia and the sol at the same time became quite clear, indicating more or less complete peptization. When the coagulated and extracted protein was boiled with water in the presence of ammonium thiocyanate there was no visible amount of peptization. The difference between this case and the case above, where the peptizing agent was added after the boiling seems to be concerned with the swelling of the albumin particles. When the electrolyte was present during boiling the amount of swelling appeared to be distinctly less than when it was added after the system was boiled and habcooled to room temperature. The effect of various reagents which could reasonably be presumed to exert a peptizing action upon coagulated egg white was next studied. The sample was prepared by diluting egg white with an equal volume of water and heating the sol so formed for I 5 minutes in boiling water. The coagulum was extracted for j 2 consecutive hours with ether. When the extraction was completed the coagulum was found to be very dry. Upon placing a portion of it in boiling water peptization occurred. A large volume of this sol was then prepared. The effect of peptizing agents was tried on separate portions of the sol. Potassium thiocyanate in an amount such that it was very concentrated in respect to the electrolyte was added to 1 5 cc. of the sol. After shaking by hand for a few minutes and allowing to stand for a day the sol was quite cloudy. The addition of a little glacial acetic acid caused considerable precipitation. This shows us that there was a significant amount of coagulated albumin peptized by the water and the electrolyte. The experiment was repeated using potassium iodide. This time the glacial acetic acid produced a very large amount of coagulation when added to the sol. When the experiment was tried using cane sugar instead of an electrolyte peptization takes place. The sol so produced does not coagulate when glacial acetic acid is added. Nitric acid does, however, cause coagulation. It has been found’ that both sulphur dioxide and formaldehyde prevent the heat coagulation of albumin sols. It was also found that this property is probably not dependent upon reduction because other reducing agents were without effect. One-half cubic centimeter of formaldehyde was added to I O cc. of the sol and the system was agitated for a short time. I n two days the sol was tested for peptized protein by adding acetic acid. There was no precipitation. However, nitric acid caused a noticeable amount of coagulation. Gortnerz states Munaretto: Arch. farm. sper., 14, 460, *“Outlines of Biochemiatry,” 404 (1929).

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WILDER D. BANCROFT AND J. E. RCTZLER, JR.

that formaldehyde combines with the proteins, whereas there is apparently no evidence, in the case of egg white, that the action is anything other than a peptization. Because of the utility of a good chemical test for albumin at this stage of the work the sodium nitroprusside (Na2Fe(CN)5?r'0.gH20)test' was examined critically. The test is stat'ed to be for the presence of an SHgroup. I t is performed by adding to one to two cubic centimeters of the substance to be tested an excess of finely powdered ammonium sulphate. This is followed by two to four drops of a freshly prepared j% sodium nitroprusside solution. The system is made alkaline by the addition of a few drops of ammonium hydroxide. A magenta color indicates the presence of denatured protein. The author states that crystallized egg albumin gives no reaction when this test is applied but that albumin coagulated by heat or acetic acid gives the test immediately. Egg white evaporated on an extensive surface showed only a doubtful nitroprusside reaction, when tried by Harriss. When dried egg white was suspended in water it was found to give a distinctly positive reaction upon performing this test'. The white of an egg was diluted to one half with distilled water and coagulated by heating in boiling water for 14minutes. The gel was cooled and the nitroprusside reaction tried on a portion of it. No reaction was noticeable. These experiments were repeated with the same results as before. The experiments were then repeated twice again, allowing more time for the reaction to take place and varying the amount of sodium nitroprusside solution used. The results were no different this time. Finally the test was carried out on a sodium thiosulfate solution which was acidified with a little acetic acid. A magentacolored ring formed where the ammonium hydroxide came in contact with the body of the liquid. From this it would appear that colloidal sulphur also gives the nitroprusside test for denaturation. These experiments indicate that this test is not to be regarded as reliable enough for our use in studying heat coagulation, for it may fail to work when it should, and it may be positive when it should be negative. The effect of dextrose upon heat coagulation was studied briefly. A 15:k egg white sol was not prevented from coagulating by the addition of 0 . 2 5 gm of dextrose to I O cubic centimeters of the sol. However, when the sol was saturated with respect to dextrose heating in boiling water caused no coagulation, Considering this from the colloidal viewpoint it offers an explanation for the peptizing action of cane sugar upon coagulated albumin. We shall see later just how this ties in. Having repeptized coagulated egg white, and, further, having prevented the coagulation of egg white sols it now only remains to be shown that the repeptized sols are chemically identical with the original sol. For this purpose the immuno-biological reaction known as the precipitin reaction was chosen.* Harriss: Proc. Roy. Soc., 94B, 426 (1923). Our thanks are gratefully extended to Profeasor R. A. Hagan, Head of the Department of Veterinary Bacteriology of the New York State Veterinary College a t Cornell University for his supervision of and help with this part of the work. 1 2


’57

The protein precipitins are highly specific, They show their characteristic reaction only with the proteins which were used in preparing them.’ Investigating the effect of chemical and physico-chemical changes of the protein molecule upon its antigenic behavior, Obermayer and Pick2 state that both types of change prevent its visible reaction with native immune serum. Pick3 came to the conclusion that agencies which effect, even in the slightest degree, the properties of the proteins lessen or completely prevent their antigenic behavior. Anson and Mirsky4 found that upon reversing denatured hemoglobin the specificity, which was lost in the denatured protein, is restored in the regenerated product. Jordan5 says: “Particular groups within the protein molecule-the aminoacids-are probably responsible for the specificity of anaphylaxis and other antigen-antibody reactions. Both chemical composition and isomeric arrangement doubtless play a part in the close correspondence that exists between antigen and antibody.” The precipitin reaction, therefore, is an extremely sensitive test for establishing the chemical identity of coagulated, repeptized albumin with native albumin. Fresh egg white was diluted with distilled water to form a 15% egg white sol. The system was agitated for less than ten seconds under a high speed soda mixer to homogenize it. Globulins precipitated out. They were removed by passing the sol through coarse filter paper three times. It was allowed to stand for two days, during which time incipient precipitation occurred, and then it was filtered three more times using ashless filter paper. The sol so prepared was passed through a sterilized Berkefeld filter. Five cubic centimeters of the sterile sol was injected intravenously into each of two rabbits. One week later ten cubic centimeters of the sol, which had been kept at 401 was injected into each rabbit. After a lapse of two weeks ten cubic centimeters of a freshly prepared sol was again injected into each rabbit. Finally, five days later the rabbits were injected with the third ten cubic centimeter dose, this time subcutaneously. Ten days were allowed for the serum to build up its titer. The rabbits were then bled, the blood centrifuged, and the serum recovered. This serum was used for the precipitin reaction. Two methods for repeptizing heat-coagulated egg white were used in preparing the antigen for the precipitin reaction. The white of an egg was diluted with distilled water to make a soyo homogeneous egg white sol. The system was heated in boiling water for 1 5 minutes to coagulate the protein. It was brought to room temperature and 85 cc. of distilled water added. Chemically pure urea was added to the system until it was practically sat-

’ Spiegel-Adolf: Alexander’s “Colloid Chemistry,” 2, 31 I (1928).

* Wien. Klin. Rundschr., 1902,No. 15;Wien. Klin. Wochenschr., 1903,659;1906,No. 1 2 . Beitr. Chem. Physiol. Path., 1, 445 (1902.) J. Gen. Physiol., 9, 169 (1925). “A Textbook of General Bacteriology” 193 (1929).

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WILDER D. BANCROFT A N D J. E. RUTZLER, JR.

urated. The large test tube containing the system was shaken moderately for 2 4 hours with the addition of small amounts of urea from time to time. The sol so formed was cloudy. A portion of it was dialyzed against running distilled water using a small parchment dialyzing thimble. The size of the dialysate container and the rate of flow of the water were so arranged that there was the equivalent of a complete change of the water every 20 to 30 minutes. The sol was dialyzed for three days; a t the end of that time the dialysate showed no residue upon evaporation at room temperature under a vacuum. The sol so produced was cloudy, but contained invisible particles as proved by the precipitation with acetic acid. The other sample was prepared from a 50% egg white sol as above. Ether, to the extent of three times the volume of the coagulum, was poured on top of the cooled coagulurn. The system was allowed to stand for a couple of hours in contact with the ether. The ether was poured off and another portion added; this was also allowed to stand for two hours. Immediately upon pouring off this second portion of the ether, part of the coagulum was put into distilled water and heated on a boiling water bath. This treatment drove off the ether and at the same time mechanically disintegrated the albumin particles; the expanding ether probably burst the particles. The albumin then began to imbibe water and further reduce in size, The water sheath is thus, a t least in part, restored to the particles, causing them to stay suspended. Potassium thiocyanate was then added to the sol until it was almost saturated. This was accompanied by a distinctly visible decrease in the cloudiness of the sol. At the same time there was no precipitation of the colloid particles. The clearing up was due, therefore, to further peptization of the albumin, by the thiocyanate ion. This sol was dialyzed in the same manner as the one previously described. Again there was a significant amount of albumin in suspension as proved by its precipitation upon the addition of acetic acid. At the end of three days neither the sol nor the dialysate contained the thiocyanate ion as proved by the absence of the red color upon bhe addition of ferric chloride solution, Both of the sols just described formed an absolutely clear colloid suspension when diluted one to ten with physiological salt solution. The precipitin reaction was studied by adding the undiluted serum t o portions of the two sols which were diluted with physiological salt solution. Approximately 0 . 2 cc. of the rabbit serum was layered against an equal amount of the antigen in its various dilutions. The following tabulation shows the results that were obtained.

TABLE I Dilution of Antigen 1;10

1;1oo

I ;Io00

Reaction of CO(NHd9 Repeptiaed sol

+ + + 0

Reaction of KCNS Repeptiaed sol

Reaction of untreated 15% egg white sol

+ + + + + + + + + + + +

0.85%

NaCl Solution 0


159

From the data obtained from this reaction it seems quite obvious that urea and potassium thiocyanate both repeptize heat-coagulated egg albumin. Furthermore, it appears certain from these tests that the heat-coagulated and repeptized albumin is chemically no different from the original material. That the precipitin reaction is extremely specific is also shown by the results of a test performed on egg albumin which was coagulated by alcohol and repeptized by concentrated potassium thiocyanate and six drops of Nizo sodium hydroxide per 40 cc of the sol. Although it was evident that there was repeptization, the dialyzed sol showed no precipitin reaction. This, in addition to what others have found, apparently established beyond a doubt that the test as used here, for this specific purpose, using as antigen and precipitin those things which were used, and only those things, is extremely highly specific, The case of alcohol coagulation will be considered later on. Although the whole white of an egg was used in this work, there can be no question about whether the globulin was responsible for the precipitation or not. The simple answer to that is that there was no globulin present. Globulins are insoluble’ or more correctly, form such unstable sols in pure water that they will, as we have seen, precipitate out of an egg white sol upon merely diluting2 it with distilled water. Ostwald finds that a 10% egg white sol in distilled water contains practically no globulin. Since native egg white sols that do not contain too large an amount of the disperse phase stay in suspension a t the isoelectric point, the claim is made that to prove the identity of the repeptized protein with the native material, it too should not precipitate when brought slowly to the isoelectric point. Quite obviously the native albumin, which stays up a t the isoelectric point, is the abnormal case; whereas, the repeptized material which comes down a t the isoelectric point acts as it should. Repeptization of heat-coagulated egg-white sols, using ether to produce mechanibal disintegration, gives us a sol which does not precipitate completely when it is brought slowly to the isoelectric point. When the experiment is not carried out correctly, however, the protein precipitates quantitatively upon bringing the sol to the isoelectric point. I n other words, the system protein-ether should undergo a sudden and large temperature change when it is immersed in hot water. Thus, we have advanced further proof that the coagulation of an egg white sol is a reversible physical change, if one is willing to believe that similarity of behavior at the isoelectric point proves anything about chemical constitution. Many heat-coagulated egg-white sols that are repeptized do not stay up when they are brought slowly to the isoelectric point. The apparent reason for this is that the initial heating destroys the action of some protecting colloid that is present in the original system. This principle is illustrated by the following experiment. When enough Congo Red sol is added to a repeptized Ostwald: “Practical Colloid Chemistry,” 159 (1924); Sumner: “Bio logical Chemistry,’ 86 (1929); Lloyd: “Chemistry of the Proteins,” 44 (1926). Ostwald: “Practical Colloid Chemistry,” 157 (1924).

I 60


albumin sol that would itself precipitate at the isoelectric point, the albumin does not all come down when the system is brought slowly to a pH of 4.8, the isoelectric point of egg albumin. These experiments prove, therefore, that heat coagulation is strictly a reversible change. How the reversible change can be chemical rather than physical is not obvious. For instance, how potassium thiocyanate, urea, potassium iodide, sodium hydroxide, cane sugar, formaldehyde and ammonium phosphate, in the case of egg albumin, potassium thiocyanate, sodium salicyl;tte, sodium hydroxide, and sodium benzoate, in the case of serum albumin, could each cause the reversal of the same chemical change (were heat dcnaturation a chemical change) is not nt all apparent. These compounds possess no characteristic which is common to all of them; some are alkaline in water solution; while others are neutral; some are electrolytes while others are not; some are reducing agents and others are not. Yet, this divers array of compounds all cause albumin of one kind or another to assume its original form after heat coagulation. A4nsonand Mirsky’ have said that the denaturation of proteins is a truly reversible process, but it is ordinarily masked by the flocculation of the denatured protein. Had the authors inserted thc word colloidal, their statement would have completely covered the case as it apparently is. So-called denaturation has been shown to be a colloidal phenomenon and therefore responsible to the rules of colloid chemistry. The actual mechanism of heat coagulation as well as other types of coagulation of this protein, being somewhat more involved than the mechanism of peptization of the coagulated protein, will be discussed at length in another paper. If, then, one is willing not t o be mystified by the word protein, the word “denaturation” should go by the boards; for we already have in the nomenclature of colloid chemistry terms which describe the process accurately. The conclusions to be drawn from this paper are: 1.--“Denaturation” has been assumed to be a chemical change. 2.-A satisfactory theory has not been offered for this change. 3 .-Other workers have retarded, prevented, and reversed “denaturation.” 4.-The problem has not, up to this time, been considered from a strictly colloid-chemical view-point. S.-Regarding egg albumin as a hydrophilic colloid, the problem to peptize heat-coagulated egg-white sols has been undertaken and solved. 6.-Potassium iodide, potassium thiocyanate, urea, ammonium thiocyanate and sodium bicarbonate, formaldehyde, and cane sugar all peptize heat-coagulated egg-white sols. 7 .-Sodium hydroxide was found to peptize heat-coagulated albumin, although there is some possibility that a slight amount of hydrolysis occurred. %-Ether does not denature albumin under ordinary conditions. J. Gen. Physiol., 9, 169 (1925).


161

g.-Egg-white sols were prevented from coagulating by extracting them with ether. Io.-It is difficult to see how ether could prevent any conceivable chemical reaction that might take place upon heat coagulation. I I .--Therefore, ether prevents “denaturation” by some sort of colloidal mechanism. 12.-The ether probably extracts something from the sol, thus giving it heat stability. 13.-The extracted material was studied as a colloid. 14.-It was shown how it can act as a coagulating agent. 15.-The substance acted like crude lecithin from egg yolk. 16.--“Denaturation” by means of ether, which does occur under some circumstances, is due merely to the removal of adsorbed water from the colloid. I 7.-Coagulated egg white repeptizes in boiling water, specially when it contains some ether to disintegrate the particles mechanically. I 8.-The presence of electrolytes during heating apparently prevents swelling. ~g.-The sodium nitroprusside test for “denatured” egg albumin was found to be wanting. 20.-Dextrose prevents heat coagulation when present in large enough amounts. z I .-Immuno-biological tests for species specificity have shown that coagulated and repeptized egg-white sols are identical with the original, as also have isoelectric point measurements. zz.--A mechanism of peptization has been proposed which initially involves mechanical disintegration of the protein coagulum by hot water, aided in some cases by ether, and followed by further disintegration and peptization by the negative ions of various electrolytes. ns.--The dogma of denaturation has been deleted. Cornell rnzcersotu.

c

The Denaturation of Albumin

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