THE EFFECT OF SUGARS AND MANNITDL UPON COAGULATION OF EGG ALBUMEN THESIS FOR THE DEGREE 0591‘ S. Willard J. Duddles 1932 . I 7 ‘9.\'\Ii0li.\“"'l . Wegyzewzda t , E§K1§§FJ§J~E§flu nggwigu§.a,lwa .n.u..a...... ..u A... ..- J. .W. L... x ..J. ( riff; .t! ....H ....m a.” 4.... .1. I , ... | h. “v.1 .n. , - uni 44.3.”; 0... N43,. ..lhha Ivy”... “ml“..aham 34., 1?... .l: .....an u..'.|.. .. . THE EFFECT OF SUGARS AND MANNITOL UPON COAGULATION 0F EGG ALBUMIN A THESIS SUBMITTED TO THE FACULTY OF MICHIGAN STATE COLLEGE OF AGRICULTURE AND APPLIED SCIENCE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF.MASTER OF SCIENCE By dILLABD J; DUDDLES June, 1932 ACKNOWLEDGMENT I wish to express my gratitude for the helpful suggestions and kindness extended to me by Professor 0. D. Ball. INTRODUCTION A study of the coagulation of proteins is important from a biological standpoint because of the importance of proteins in life processes. As far as is known proteins are always present in living tissues and play an important part in many of the vital reactions taking place there. Coagulation changes the preperties of proteins to such an extent that their functions must be impaired. It is known that death of living tissues is accompanied by a coagulation of the proteins present. It is possible that this is the cause of death in some cases. In choosing a protein suitable for a study of the coagulation process, it is desirable to use one which can be easily coagulated by heating. Egg albumin is found to have this characteristic as well as the advantage of being oomparitively easy to obtain in a pure form. It is one of the few proteins which will crystallize, thus making purification relatively easy. The fact that egg albumin is important in its biological functions also makes its use in experimental work more desirable. DEFINITION OF TERMS In reviewing the literature on proteins, there is found a confusion of the terms denaturation, coagulation, and flocculation. Sorensen uses these terms in accordance with the following definitions: Denaturation is a change of a kind not yet under- stood which native protein is capable of undergoing in the presence of water. This change is not necessarily accompanied by precipitation of the protein from the solution. Flocculation is the precipitation of the protein in an amorphous form. This precipitation is possible only after the protein has undergone the change known as denaturation. The two consecutive changes-~namely, denaturation and flocculation--taken together are known as coagulation. Throughout this paper I shall use the terms in accordance with these definitions. HISTORICAL In treating the historical aspect of this problem, I wish to consider the following subjects: 1. Whether protein denaturation is of a hydrolytic or dehydrating nature. 2. Liberation of sulphhydryl groups during denaturation. 3. The possibilities of reversing the denaturation re- action. 4. The acid and base binding power of proteins before and after denaturation. 5. The possible liberation of ammonia or other nitrogenous compounds upon denaturation. 6. Factors influencing the denaturation reaction. WHETHER PROTE N DENATURATION IS OF A HYDROLXTIC OR DEHYDRATING NATURE Some of the earliest work done to determine whether or not the process of denaturation of proteins was accompanied by a gain or loss of water was by T.B. Robertson. (l)_ Robertson, working with casein, observed that the power of the bases, potassium hydroxide and lithium hydroxide, to dissolve casein was greatly increased by increasing the temperature. In all cases the above results were obtained where the solutions were acid to phenOlphthalein and approximately neutral to litmus, so that the effect of heat— ing the solution of caseinate could not be to increase its hydrolytic dissociation. Robertson eXplained the greater solubility with temperature increase as being due to a shift of the equilibrium of the following equation to the right. EXOH + HXOH=HXXOH * H20 Thus a given amount of alkali, since it was associated with a molecule of nearly double weight, neutralized twice the number of equivalents of casein at 66°C. as it neutralized at room temperature. He held the opinion that in the case of heat coaguable proteins, the flocculation was probably due to repeated condensations of the type above, to form larger aggregates. H. Chick and C. J. Martin (2) observed that dry, crystalline egg albumin could be heated in a hot air bath at 105°C. for five hours and still be completely soluble in water. Samples of egg-albumin crystals identical with the ones heated dry were heated at 105°C. in the presence of steam for five hours and found to be completely insoluble at the end of that time. They considered these results to show conclusively that heat denaturation is a reaction be- tween the protein and water. This property of denaturation had been observed by A. Wichmann in 1899 (5) and has since been observed in vegetable proteins by T. B. Osborne (4). Osborne found that vegetable proteins boiled in alcohol were not denatured, while samples of the same protein heated to the same temperature in.water were denatured. Chick and Martin attempted to show that the denaturation reaction was of a hydrolytic nature. They ran formol titrations before and after denaturation but their results gave no significant information. The same authors (4) determined the rate of the denaturation reaction and its relation to the concentration of protein. These results showed that the rate of de- naturation by heat was directly pr0portional to the con- centration of the reacting substances, protein and water. The amount of water which entered into the reaction was so small compared to the amount of water present, that it was disregarded. Chick and Martin considered that this result showed conclusively that the denaturation by heat was a reactiOn of the first order, a monomolecular one. w. w. Lepeechkin (5) in reviewing the work of Chick and.Martin on the effect of hydrogen ion concentration on heat denaturation rate and in view of his own experiments, concluded that proteins undergo a slight hydrolysis upon being denatured by heat. The fact that denaturation of proteins was greatly influenced by hydrogen ion concentration and that many hydrolytic reactions, such as inversion of sucrose, were also greatly influenced by hydrogen ion con- centration, led him to assume that the reactions were similar. He attributed the failure of Chick and Martin to obtain evidence of hydrolysis, to the fact that the protein mole- cule was large and the amount of hydrolysis slight. The next important piece of work done on this particular phase of protein denaturation was that of S. P. L. Sdrensen (6). Surensen used indirect methods for determining the amount of water in the protein molecule before and after denaturation. He detennined the amount of ammonium sulphate bound by undenatured egg-albumin when crystallized out of ammonium sulphate solution of known concentration. He then determined the amount of ammonium sulphate bound by heat denatured albumin when it was floc- culated from an ammonium sulphate solution of equal con- centration. Considering the amount of ammonium sulphate bound, to be a measure of the amount of water contained in the protein molecule, the denatured protein contained less water than the undenatured protein. He repeated the experiment using alcohol as the denaturant and again the denatured protein bound less ammonium sulphate than the undenatured. Sorensen's conclusions were that egg-albumin lost water when denatured by heat or alcohol. He concluded that the egg albumin always retains some water. H. Wu and_D. Y. Wu (7) presented evidence which they considered indicated that heat denaturation was a hydrolytic reaction. This view was based upon the fact that egg albumin, upon undergoing heat denaturation, increased its chromogenic value toward Folin and Dennis phenol reageant and also increased its acid and base binding capacity. Proteins upon undergoing hydrolysis also increased in these pr0perties. Therefore they concluded that denaturation must be hydrolytic in nature. They also concluded that since the filtrate from heat coagulated proteins contained more nitrogen than the filtrate from.samples of the pro- tein solution precipitated in the cold, that some amino acids were Split off during heat denaturation. w. M. 0. Lewis (8) in reviewing the work of Chick and Martin, expresses the Opinion that protein denaturation.may be hydrolysis of some of the peptid linkages, accompanied by an equivalent amount of closing of other linkages by loss of water. He also suggests that this imay be accompanied by a physical distention of the molecule.” LIBERATION OF SULPHHYDRYL GROUPS DURING DENATURATION It is a fairly well established fact that egg albumin contains cystine in its molecular make-up. This amino acid almost certainly represents the form in which most of the sulphur is combined in egg albumin and probably in other proteins. 9. Embden (9) was an early worker in searching for further evidence as to the form of sulphur found in proteins.‘ He claimed that cysteine was a constituent of proteins as well as cystine. He isolated cysteine from the products of hydrolyzed egg albumin. K. A. H. Hdrner (10) claimed that Embden had in reality subjected his protein to such treatment that some of the cystdhe ‘would be reduced to cysteine during hydrolysis. This would account for the cysteine isolated by Embden. By quantitative methods of analysis based on the blackening of lead acetate Mdrner (lO) concluded that all the sulphur in albumin and several other proteins was in the form of cysteine. His methods were rough and unp certain, however, as was pointed out by L.J. Harris (14). T. B. Johnson (ll) suggested the possibility of a thiopolypeptide linkage which might yield sulphhydryl groups upon hydrolysis. V. Arnold (12) applied a nitroprusside test to several proteins. This test consisted of the addition of an excess of ammonium sulphate to one or two cos. of the substance to be tested, then adding two to four drops of sodium nitrOprusside and a few drops of ammonium hydroxide. He observed that tissue extracts and certain coagulated proteins gave a characteristic purple color with this test. He attributed this reaction to the presence of cysteine which contains a sulphhydryl grouping. S. B. Osborne and H. H. Guest (13) observed that casein decomposed in strong alkalies yielded a very much smaller proportion of sulphide-sulpher than did the other proteins. This led them to the conclusion that most of the sulphpr was present in some other form than cysteine. L. J. Harris (14) observed that undenatured egg albumin would not give the sodium nitroprusside test, while heat denatured egg albumin gave a very strong purple color in response to the test. He also observed that egg albumin crystalized from ammonium sulphate failed to give the test while egg albumin crystalized, redissolved, and heated did give positive results. Further observation by Harris showed that egg albumin precipitated with weak alcohol, and which could be redissolved, gave negative results, However, egg albumin coagulated by strong alcohol or hydrochloric acid gave a positive test with sodium nitrOprusside. Upon exposure of egg albumin to ultraviolet light, Harris observed that a light, feathery precipitate was obtained. The precipitate he found was soluble in water. This fact had also been noted by E. G. Young (15). Harris found that this soluble precipitate would not give a nitroprusside test. Upon further exposure to ultraviolet a stringy coagulum was produced which yielded a faint purple color with sodium nitrOprusside. Theories advanced by Harris to account for this appearance of reactivity to sodium nitrOprussido upon de- naturation of proteins are as follows: first, the possibility of a thicpeptide linkage, which underwent hydrolysis upon denaturation, producing a sulphydryl group; second, the possibility of a linkage of the R - C’O’S - R type which upon hydration would yield the sulphydryl grouping; third, the replacement of a carboxyl group by a sulphydryl function- ing acidically; fourth, a tautomeric change without assuming that denaturation involves hydrolysis. C. Rimlngton (1‘) stated that wool, when treated with ultraviolet or alkalies, yielded a positive sodium nitroprusside test. REYERSAL OF DENATURATION Perhaps the earliest data which would indicate the possibility of reversal of denaturation was that presented by Corin and Ansiaux (17). These workers observed that if, during the flocculation of heat de- natured egg albumin, the mixture were shaken vigorously upon the very first appearance of a precipitate,the pre- cipitate would disappear temporarily. Among the early workers who advanced the idea of the possibility of reversing the denaturation reaction of proteins, were L..Michaelis and P. Rona (18). They observed the fact that, when serum albumins were coagulated rapidly by heat, the resulting eoagulum was somewhat soluble in water, while the coagulum obtained by heating slowly was entirely insoluble. Adolf (56) found that by’dissolving coagulated serum albumin in dilute sodium hydroxide and removing the alkali by dialysis, that an albumin corresponding to the original sample was obtained. This regenerated albumin had the same hydrogen ion concentration, specific electrical conductivity, Optical rotation, temperature of coagulation and protective value toward gold and mastic sols, as the original albumin. Perhaps the most important piece of work done regarding reversibility of proteins was that of m. L. Anson 10 and A. E.llirsky (19). They found it possible to prepare from heat denatured hemoglobin a hemoglobin soluble at the isoelectric point. They denatured a sample of hemoglobin by heating it in a slightly acid solution at 80°C. for three and one half minutes. While the solution was still hot it was brought to about pH9 by addition of a Potassium cyanide - hydrochloric acid buffer. After cooling, the solution was adjusted to the isoelectric point and filtered. The filtrate was saturated with carbon monoxide and reduced with sodium hydrosulfite. The supposedly reversed hemoglobin was then precipitated from the filtrate by addition of ammonium sulphate. About thirty percent of the original hemoglobin was thus obtained. According to Anson and Mirsky this reversed hemoglobin was capable of undergoing heat coagulation, could be crystallized out of solution by addition of ammonium sul- phate, yielding crystals of the same shape as undenatured hemoglobin, and had the same color as the original hemoglobin. It also gave the same absorption spectrum and had the same affinities for oxygen and carbon monoxide as the original hemoglobin. In a later work Anson and Mirsky (20) had improved their technique and procedure to the extent that seventy- five percent of the original sample could be recovered as reversed hemoglobin. In a review of their work on reversing the de- naturation reaction,.M. L. Anson and A. E. Mirsky (21) 11 claim to have reversed proteins other than hemoglobin from the denatured state to their original form. Among these proteins are serumglobin and serum albumin. As yet they have been unable to prepare native egg albumin from coagulated egg albumin. At about the same time that Anson and Mirsky were working on reversibility of denaturation, w. D. Bancroft and J. E. Butzler, Jr. (22) claimed to have ob- tained similar results with egg albumin. Their procedure was entirely different from Anson and.Mirsky's, however. They prevented heat coagulation of egg albumin by extraction of the egg ablumin crystals with other. They also claimed to have repeptized coagulated egg albumin by extracting the coagulate with ether and shaking in an ammonium thiocyanate solution. Similar results were obtained by using potassium iodide instead of the thiocyanate. Nothing is mentioned in their work about the pH of the solutions in which these re- sults are obtained. They also claimed to have dispersed coagulated egg albumin by saturation with sucrose or with glucose. Their view is that coagulation is only an aggre- gation of the protein molecules which can be rediSpersed by the above methods. The reaction preceding flocculation, commonly known as denaturation, is not recognized by them. 12 ACID AND BASE BINDING POJBR OF PROTEINS BEFORE AND AFTER DENATURATION The fact that free acidity decreased when proteins were coagulated from an acid solution, and that free alkalinity decreased when they were coagulated from alkaline solutions was first observed by N. D. Halliburton (23). The same behavior of proteins upon coagulation was observed by J. B. Haycraft and C. N. Duggan ( 24). They suggested that this behavior might account for the continual "raising of coagulation temperature" during the process of coagulation. H. Chick and C. J. Martin (2) made further investi- gations on the increase in acid binding power of proteins upon denaturation. Their procedure involved the measurement of hydrogen ion concentration by electrometric methods, before and after denaturation in an acid solution. They found that for small concentrations of acid, up to .0001 N., the amount of hydrogen ions withdrawn from the solution was nearly proportional to the initial concentration of free acid. Above this strength the acid removed became pr0portionally less. H. Wu and D. Y. Wu (7) demonstrated the increase in acid and base binding power by merely heating test tubes of egg albumin plus acid or alkali and noting their change of pH by use of indicators. B. M. Hendrix and V. Wilson (25) obtained results entirely Opposite from those of Chick and Martin. They found 15 that undenatured egg albumin required more acid to adjust it to a given pH than did the same amount of denatured egg albumin. Their procedure involved heating the egg albumin for thirty minutes at a pH of 4.7. The protein was then allowed to stand over night before the titrations were per- formed. P. S. Lewis (26) found no difference in the acid and base binding power of denatured hemoglobin and undenatured hemoglobin. H. du (27) claims that denaturation is a hydrolytic fission of the protein molecule. He claims that flocculation is a molecular condensation of these hydrolyzed molecules. This he believes accounts for the increased acid and base binding power of denatured proteins. The work of Lewis was repeated by N. Booth (28) who used egg albumin instead of hemglobin. He also found no difference in the acid and base binding power of denatured and undenatured egg albumin. Booth claimed that the work of Lewis was not reliable owing to the fact that hemoglobin was not stable at the pH at which several of Lewis's titrations were carried out. Egg albumin being more stable than hemoglobin, Booth considered his own work to be more conclusive. 14 THE POSSIBLE LIBERATION OF AMMONIA OR OTHER NITROGENOUS COMPOUNDS UPON DENATURATION S. P. L. Syrensen and E. Jurgensen (29) attempted to determine whether or not denaturation of proteins involved liberation of ammonia. They found that, upon heating egg albumin at or near its isoelectric point, the formol- titratable nitrogen in samples of filtrate continued to in- crease steadily with the time of heating, whether floccula- tion was complete or not. This is fairly conclusive evidence that the coagulated albumin undergoes some hydrolysis in the hot water after flocculation. Surensen found no significant increase in formol-titratable nitrogen above that which was accounted for by decomposition of denatured albumin. Later experiments by Surensen.(.6) were performed, employing the same technique but using a purer egg albumin prepared by repeated crystalizations with ammonium sulphate, freed from ammonia by washing with potassium or sodium sulphates and recrystallized using these salts as precipitants. This albumin had the advantage of having the entire amount of nitrogen present coaguable. Using this albumin it was observed that decomposition of the denatured albumin was least in samples near the ido- electric point of the protein and greatest in samples furthest removed from the isoelectric point. The results are eXplained by Surensen as being due to the action of the hot water on the denatured, unprecipitated protein, bringing about decomposition. His conclusion is 15 that the denaturing reaction itself does not liberate ammonia or other nitrogenous substances. To further prove this point Sorensen repeated the experiment using alcohol as the denaturing factor rather than heat. He failed to find significant amounts of ammonia or nitrogenous material in the filtrates from the coagulated albumin. FACTORS INFLUENCING PROTEIN DENATURATION The effect of acids and bases upon the rate of denaturation of proteins by heat was first observed by w. D. Halliburton (85). He observed that in solutions alkaline to litmus the temperature at which precipitation occurred was not much influenced, if at all, by the degree of alkalinity. 0n the other hand the addition of acid lowered the teuperature of coagulation. H. Chick and C. J. Martin (2) found that alkali- albumin was formed in solutions of hydroxyl ion concentration twice that of distilled water. They made a careful study of the effect of acid upon rate of denaturation. Their re~ sults confirmed those of Halliburton and of Haycraft and Duggan. Chick and Martin (51) made the observation that salts, up to a certain concentration, decreased the rate of denaturation of egg albumin by heating. This protective action was noticed up to a concentration of three normal in the case of sodium chloride and ammonium sulphate. They also noticed that when neutral salts were added to acid pro- 16 tein solutions a decrease in free acid occurred. In 1912 Chick and Martin (52) tested the effect of alkali upon the rate of heat denaturation of egg ablumin. They found that an increase in hydroxyl ion concentration increased the rate of denaturation Just as an increase in hydrogen ions had affected it on the acid side of the iso- electric point. In all cases the rate of denaturation was proportional to the concentration of unchanged albumin, if the pH of the solution were kept constant during the de- naturation. A. Beilinnson (55) found that sucrose inhibited coagulation of proteins by heat. By complete saturation of serum albumin with sucrose it was made thermostable at 62°C. Egg albumin was found to be thermostable at 75°C. when saturated with sucrose. The protein solutions were heated at pH 7.5 - 7.6 and then adjusted to near their iso- electric points. The amount of denaturation was measured by titrating to standard turbidity with.saturated ammonium sulphate solution and checked by nitrogen determinations on the precipitate. Glycerol was found to have a definite stabiliz- ing effect against heat denaturation. The protection was not nearly so complete as with sucrose. Another piece of interesting work was done by Torsten Teorell (84) who found that the formation of the coagulate in heat denatured serum albumin was inhibited by alcohol in the presence of an acetate buffer. The inhibition increased with an increase in concentration of the acetate buffer. l7 Propyl alcohol showed a greater inhibiting effect than ethyl alcohol and ethyl greater than methyl alcohol. The coagulate appeared to dissolve in hot alcohol of less than 20% concentration. It precipitated out again as the alcohol cooled. Serum albumin and native plasma of horse behaved as described above, but ovalbumin was not affected by the presence of alcohol. Ihile the present work was in progress, R. Newton and w. R. Brown (55), in working with plant saps, found that sucrose and glucose prevented denaturation of plant proteins by freezing. They found that by adding the sugars to the unaltered plant sap a definite protection against coagulation by freezing was shown, up to a concentration of 8% sugar. After that concentration was reached, additional sugar had little or no effect. EXPERIMENTAL A solution of egg albumin prepared according to Scrensen's method (58) was used in eXperiments to determine the effect of sugars upon protein coagulation. The nitro- gen content of the albumin solution was determined in duplicate by a.modified micro-Kieldahl method using methyl red as an indicator (59). Titrations were made with .01 N. sulphuric acid. The effect of glucose upon heat coagulation of egg albumin was studied in the following manner. A solution of egg albumin containing 1.5 mgs. of nitrOgen per cc. and sufficient sodium acetate--acetic acid buffer of pH 4.8 to insure a constant pH of that value, was prepared. Ten ccs. EXperi- ment No 18 of this solution was placed in each of four large test tubes. To tube I was added 10 cos. of E; glucose, to tube II, 10 cos. % glucose, to tube III, 1010”“?6 glucose, and to tube 17, 10 cos. of distilled water. The series was prepared in duplicate. These tubes were placed in a water bath at 70°C. for 10 minutes, then removed and cooled to room temperature. The contents of each tube was filtered and the amount of coagulation determined by analyzing the filtrates for nitrogen. Results from this eXperiment are represented in table 1. Each value in the tables on nitrogen analysis represents a duplicate nitrogen determination. TABLE I Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from egg albumin (1.5 mgs. N. per 2 cos.) coagu- lated in glucose solutions by heating for 10 minutes at 70°C. at pH 4.8 ‘ M : M . _ f -,- glucose : It glucose ; 12% glucose E Distilled H203 A16.e Ne; % : Triage Ne : 7c,» MEBe Ne : 3 M38. : % n 2 ccs.:tota1‘in 2 ccs.;total i in 2 cos.:total filtrate : N.éfiltrate ; N. filtrate : N. filtrate; N L I e O ee ee)e ee ee e( in 2 ccsgtotalt ee ee .0 I. .0 .0 O. O. O .697 :46.4%: .565 257.5% .556 222.44: .564 224.2%: II .595 :59.5%: .478 :51.8%: .566 :24.4%: .556 :22.4%: III : .819 :54.6%: .477 :51.8%: .407 :27.l%: .585 :25.6%: IV : .745 :49.5%: .522 :54.8%: .592 :26.1%: .565 :24.5%: v - .602 :4o.o%: .417 :27.s%: .580 :25.5%: .556 :22.4%: VI : .759 :49.2%: .490 :52.6%: .406 :27.0%: ---- : --- v11 : .595 :59.6%: '.518 :54.5%: .294 :19.6%: ---- : --- VIC. 19 It will be observed that in all cases there is nearly twice as much nitrogen in the filtrates from the g‘glucose solution as in.the distilled water solution of the protein. A blank nitrogen determination was run on glucose solutions containing no albumin and correction made for nitrogen from this source. The amount of nitrogen in the glucose was .01 mgs. per 2 cc. % glucose. The first appearance of a coagulum in the % glucose solutions was always about a minute later than the first appearance of the coagulum in the distilled water solutions. The eXperiment was repeated using the same pro- cedure excepting that the pH was kept at 5.2 instead of pH 4.8. These results are given in table II. TABLE II Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from albumin (1.5 mgs. N. per 2 cos.) coagulated in glucose solutions by heating for 10 minutes at 70°C at pH 5.2 Experi- : M : M : M : - : ment No.: 2 glucose : 30 glucose : (:0 glucose :Distilled H20 : gifigs. N. : REY ;Mgs. N. : % ; Mgs. N. : % ; M35. in : % E :in 2 ccs.:total:in 2 ccs.:total:in 2 ccs.:total:in 2 ccs.:total: _, :filtrate : N. :filtrate : N. :filtrate : N. :filtrate : Nt_: I 2 1.550 :88.6 E 1.176 E 78.4: 1.148 E 76.5: 1.141 E 76.4; II : 1.558 :90.5 : 1.197 : 79.8: 1.155 : 77.0: 1.155 : 77.0: III 3 1.299 :86e0 3 1.120 : 74e6: 1e148 3 76e5: 10176 : 38e4: IV 3 10316 :87e6 : 1e141 3 76e4: , 10176 : 78e4: ’1e212 : 80e8: 20 In this case the coagulation was much less complete due to the fact that the pH at which coagulation took place was further removed from the iso electric point of egg albumin. The inhibiting effect of glucose upon coagulation is again significant, however. The effect of sucrose upon coagulation of egg albumin was studied next. The heating was carried out at pH 7 to prevent hydrolysis of the sucrose. The protein solution was then adjusted to pH 4.8, where flocculation took place. This pH was obtained by adding 5 cos. acetic acid-sodium acetate buffer of pH 4.8. The solutions were then filtered and the filtrates analyzed for nitrogen as before. The results obtained are in table III. No correction for nitrogen in the sucrose was necessary. The amount of nitrOgen in 2 ccs.i£"--l sucrose was not measurable with the 1 micro-Ejeldahl method. 21 TABLE III Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from egg albumin coagulated in sucrose solutions. Egg albumin solution (1.5 mgs. N. per 2 cos.) was heated for 15 minutes at pH 7 at 70°C. The pH of the cooled solution was then adjusted to pH 4.8 by adding 5 cos. acetic acid-sodium acetate buffer of pH 4.8 Experi- : M : M. : u; : f : ment No.: .2 sucrose :'Z sucrose : 26. sucrose :Distilled E20 : :iigsifii: 3’6 flags. N.: “,3? :higs.NT: 9,6 gugs. N.: %¥‘; :in 2 ccs.:total:in 2 ccs.:tota1:in 28cc.:tota1:in 2 ccs.:total: :filtrate : N. :filtrate : N. :filtrate: N. :filtrate : N. g 1 E .716 E 55.0; .672 E 51.6; .605 E 46.5: .588 E 45.22 II : .749 : 57.6: .785 : 60.5: .729 : 56.0: .561 : 45.2: III : .882 : 67.8: .798 : 61.5: .721 : 55.4: .700 : 55.8: IV : """" : "" : 0854 : 65e6: e729 : 56.0: e701 : 55.8: V : .827 : 65.6; .766 : 58.9: .750 : 56.1: .702 : 55.9: VI : ~868 : 66.7: .772 : 59.2: .756 : 58.1:. .714 : 54.8: The effect of fructose upon heat coagulation of egg albumin was atudied in the same manner in which glucose ‘had been studied. The results were similar to those obtained by glucose except that the protection was not as great. These results are represented in table I7. Correction was made for nitrogen from the fructose. Two cos. 4124 fructose equaled .01 mg. N. O '0 O .0 22 TABLE IV Results in terms of Mgs. nitrogen in 2 cc. samples of filtrate from egg albumin (1.5 mgs. N. per 2 cos.) coagulated in presence of fructose by heating for 10 minutes at 70°C at pH 4.8 522?;33 .Ié fructose : % fructose :gU fructose :Distilled H20 i Mgs. N. : %. : MgsT:N. : E : Mgs.NT’: TUgMEs. N7_?_fi%_- :in 2 ccs.:total:in 2 ccs.:total:in 2 ccs.:total:in 2 ccs.:total :filtrate : N. :filtrate : N. :filtrate : N. :filtrate : N. I ; .588 .: 59.2: .490 : 52.6: .448 : 29.8: .420 : 28.0:‘ II : .560 : 57.5: .481 : 52.0: .478 : 51.8: .425 : 28.5: III : .602 : 50.1: .590 : 26.0: .595 : 26.2: .525 : 21.5: IV : .518 : 54.5:, .417 : 27.8: .578 : 25.2: .556 : 22.4: V : .758 : 49.2: .490 : 52.6: .406 : 27.0: .454 : 28.9: VI : .688 : 45.8: .602 : 40.1: .490 : 52.6: .406 : 27.0: VII : .522 : 54.8: .480, : 52.0: .579 : 25.5: ~-- : ---: VIII : .499 : 55.5: .492 : 52.8: .592 : 26.1: .406 : 27.0: IX : .549 : 55.6: .478 : 51.8: .467 : 51.0: .548 T 25.2: X : .588 : 59.2: .488 : 52.5: .464 : 50.9: .540 : 22.6: the protecting sugar. table Ve O. O. O. O. O. O. O. The same experiment was repeated using mannose as The results obtained are shown in The protective effect of the mannose is less pronounced than in the case of glucose or fructose. 25 ' TABLE 7 Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from egg albumin (1.5 mgs. N. per 2 cos.) coagulated in the presence of mannose by heating for 10 minutes at 70°C at pH 4.8 : . M os ‘ M . = M : EXperi- : ann e g 2’ mannose : 20' Mannose :Distilled H20 msnt No.: : ;_ : 1 : 4g :Mgs. 14.: 95 : Mgs. 11.: 375—: 15387111.: 9?: figs—N. .: 76 : :in 2 ccs.:total:in 2 ccs.:total:in 2 ccs.:total:in 2 ccs.:total: :filtrate : N. :filtrate : N. :filtrate : N. :filtrate : N. I : .795 : 55.0: .712 : 47.4: .690 : 46.0: .644 : 42.9; 11 3 e802 3 55e43 e697 3 46e4: e700 3 46e6: e646 3 45e0l III 2 e739 3 49023 e660 3 44e03 e645 3 43e03 e595 3 59.0; IV e749 3 49e93 0674 : 44e93 0631 3 42003 e572 3 38011 Mannitol was substituted for mannose as a protecting agent. The protection in this case was slight. A distinct protective effect was evident, however, as will be seen in table 71. 24 TABLE VI Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from egg albumin (1.5 mgs. N. per 2 cos.) coagulated in the presence and absence of mannitol by heating fro 10 minutes at 70°C. at pH 4.8 EIperi- M Mannitol ment No.: 2' ; Distilled H20 ; E ligs. N. per : E figs. N. per : o E : 2 cos. filtrate: total : 2 cos. filtrate : total : 3 3 No 3 3 He 3 z : : : : I : .546 : 56.4 : .266 : 17.7 : II : .562 : 27.4 : .260 : 17.5 : III : .554 : 56.9 : .240 : 16.0 : IV 3 e562 3 3704 3 e308 3 20e5 : V : .602 : 40.1 : .274 : 18.2 : VI : .582 : 58.8 : : e254 ,3 16e9 Egg albumin was found to be completely stable to a temperature of 70°C when saturated with glucose or fructose. A saturated solution of glucose represents a concentration of approximately 4.6 molar with respect to glucose. A saturated fructose solution is approximately 5 molar with respect to fructose. The solutions of egg albumin saturated with glucose and those saturated with fructose remained perfectly clear after heating for 10 minutes at 70°C at pH 4.8. The sugar was then dialyzed out for the solution and the pH again adjusted to 4.8. No flocculation took place after removal of the sugar. Nitrogen analyses were run on samples of the filtrates of the albumin solutions after heating. The results are presented in tables VII and VIII. TABLE VII Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from egg albumin coagulated in saturated glucose solution and in absence of glucose by heating 10 minutes at 70°C at pH 4.8. Saturated Glucose f Distilled Water M880 No per 2 00803 if 3 M88. Yo per 2 0030: f filtrate : total : filtrate : total L 3 No 3 3 No 4.412 E 99.8 E .84 E 19.0 .0 O. I. O. O. O. O. 25 TABLE VIII Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from egg albumin coagulated in saturated fructose solution and in absence of fructose by heating for 10 minutes at 70°C at pH 4.8 Saturated Fructose Distilled Water Mgs. K. per 2 ccs.: %’ : Mgs. fi. per—2 ccs.: E filtrate : total : filtrate : total : L. N. : z N. : 3 3 3 : 4.53 : 99.2 : .68 : 15.1 : The effect of saturating a solution of egg albumin with mannitol was also studied. In this case the protection was not complete. Analysis for nitrogen was run on the filtrates from the heated egg albumin solutions. The results are represented in table IX. A saturated solution of mannitol is approximately .8 molar with respect to mannitol. 26 TABLE IX Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from egg albumin (4.42 mgs. N. per 2 cos.) coagulated in saturated solution of mannitol by heating for 10 minutes at 70°C at pH 4.8 Saturated Mannitol Distilled Water s. l. per 2 ccs.: %? E Mgs. N. per I ccs.: % E filtrate : total : filtrate : total : _. z N. : : N. : 1.150 : 25.7 : .45 : 9.7 : . The effect of glucose upon the coagulation of egg albumin by ultraviolet light was studied. It was observed that when egg albumin in % glucose solution was exposed to ultraviolet light, the turbidity was much less than in the check solution containing no glucose. The filtrates from the above coagulums were analyzed for nitrogen content. The amount of coagulation was not great enough to show much difference in the filtrates. The slight difference is in agreement with the difference in turbidity, however. The results are shown in table X. 27 TABLE X Results in terms of mgs. nitrogen in 2 cc. samples of filtrate from egg albumin (1.5 mgs. nitrogen per 2 cos.) coagulated fran glucose solution of pH 4.8 by exposure to ultraviolet light. Exposure was made for 15 minutes at a distance of 24 inches from a mercury quartz lamp. Experi- 3 M ment No.: ‘§ Glucose Distilled Water : : A : 2 111880 Fe 111 3 3 1.18:8. EC 111 : 3%)— 3 z 2 cos. filtrate: total : 2 ccs. filtrate: total: : z N. : : N. : 3 3 3 3 3 I 3 1.401 : 93.4 : 1.342 : 89.4 : II : 1.379 : 91.9 : 1.363 : 90.8 : III : 1.398 : 93.2 : 1.335 : 89.0 : The experiment was repeated, using egg albumin solution saturated with glucose. The glucose-saturated solutions remained perfectly clear while the check solution of egg albumin became very turbid. The solutions were allowed to stand for 24 hours after exposure to ultraviolet. The glucose solution still remained perfectly clear. The results from analysis of the filtrates are given in table XI. Exposures were made for 15 minutes at a distance of 15 inches from a mercury quartz light. The pH of the solutions was 4.8 in all cases. TABLE XI Results in terms of mgs. nitrogen per 2 cos. filtrate from egg albumin coagulated by ultraviolet from saturated glucose solution. ment No.: Saturated Glucose : Distilled water 3 3 FEES- E: Per : %— : Mgs. N. per : %. E : 2 ccs. filtrate: total : 2 cos. filtrate: total : : : H’ 3 3 N0__3 I E 2.911 E 99.7 2 2.382 2 91.5 2 II : 2.899 : 99.4 : 2.328 : 79.9 : The coagulum obtained from exposure to ultra- violet had an appearance different from that of the coagulum from eXposure to heat. The ultraviolet coagulum was light and almost crystaline in appearance while the heat coagulum was stringy and viscous. In an attempt to investigate the cause of the protective effect of sugars against protein coagulation, ”bound water” determinations were made on the glucose- albwmin solutions and on the check solutions containing no glucose. The method outlined by Gortner (37) was used in making these determinations. The results did not check closely enough to be very reliable. The average results indicated, however, that there was more water ”bound" by the albumin in glucose solution than in the absence of 30 glucose. Some of these results are presented in table XII. TABLE.XII Increase in freezing point depression indicating increase in “bound water" due to the presence of.E glucose. 2 Average depression : Average depression : Average increase in: of f.p. due to su- : f.p. due to sucrose: f.p. depression due: cross in presence : in absence of : to sucrose in pre- : Of‘% glucose : glucose : sence ofIM glucose : 2.256°C E 2.145°c E .111°c E Each of the values in this table represents an average of eight determinations. An attempt was made to repeptize the coagulum obtained by heating egg albumin at 70°C for 10 minutes at pH 4.8. The method used was to saturate the solution con- taining the coagulum with glucose. This was accomplished by adding an excess of solid glucose to the solution con- taining the coagulum and allowing it to stand for 24 hours with frequent stirring. A.check which contained no glucose was made up to the same volumn as the saturated solution with distilled water. The two solutions were then filtered and samples of the filtrate analyzed for nitrOgen. The results are given in table XIII. The increase in nitro- gen content of the glucose-saturated solution is too slight to indicate any appreciable amount of peptization of the coagulum. TABLE XIII Results from attempt to repeptize coagulated egg albumin with glucose. Filtrate from glucose-saturated: Filtrate from egg albumin: solution in absence of glucose egg albumin solution Mgs. N. per 2 cc. samples Mgs. N. per 2 cc. O. I. O. O. O. O. I. filtrate samples filtrate; .968 .952 .958 : .949 31 32 DISCUSSION The results of the coagulation experiments indicate that the sugars glucose, fructose, mannose and sucrose as well as mannitol have a definite inhibiting effect upon coagulation of egg albumin by heat or by ultraviolet light. The protective effect of glucose and fructose is nearly equal, while that of mannose and mannitol is somewhat less. The amount of protection caused by the presence of sucrose is not comparable to the amount caused by the other sugars or mannitol because of the higher pH at Which heating was necessarily carried out. The fact that no coagulation took place when albumin was heated at its isoelectric point in saturated glucose or fructose solutions was significant. Further- more, the fact that no flocculation took place after the sugar was removed by dialysis indicates that the sugar prevented the denaturation reaction from taking place, not merely the flocculation. The work of Newton and Brown (35) is closely related to the protective effect of sugars shown in the results of this thesis. They worked with native plant 'sap while the present work was with egg albumin, a definite protein. They found that sucrose and glucose prevented the coagulation of nitrogenous material from the sap by freezing. At least part of this nitrogenous 33 material was undoubtedly plant protein. The sugars evidently prevented the coagulation of the plant proteins by freezing Just as they have been found to prevent egg albumin from coagulation by heat. Newton and Martin found that above a sugar concentration of 8%,little or no decrease in amount of material coagulated was observed. This may have been due to the fact that a certain amount of the plant nitrogenous matter is not coaguable by heat. In the present work the protective effect of glucose against coagulation of egg albumin by heat in- creased until complete protection was afforded when the saturation pcint was reached. The wont of Beillinson (33) is also closely related to the results obtained in the present work. He obtained protection of serum albumin and egg albumin against heat coagulation,by addition of sucrose or glycerol. He was able to make serum albumin and egg albumin completely stable to a temperature of 62°C and 75°C respectively, by saturating the protein solutions with sucrose. The reason for the protection against coagulation afforded by the presence of sugar is not easily explained. The fact that there is an indication of more "bound water" in a solution of egg albumin ini% glucose solution than by a solution of equal protein concentration in water, suggests the possibility of water being withheld from the denaturation reaction by the sugar. The same fact may be interpreted to indicate an increase in water "bound" by the protein itself. 34 If, as Sorensen (6) suggests, protein denaturation in- volves a loss of water by the protein, the fact that more water was bound by the protein would inhibit de- naturation. An attemptwas made to determine whether or not denaturation of egg albumin by heat was accompanied by an increase or decrease in the amount of water "bound“. The change in the amount of "bound water" before and after heating was so slight that in order to obtain a measurable difference a concentrated protein solution was necessarily used. In using the more concentrated protein solution, it was impo ssible to obtain satisfactory results due to excessive flocculation of the protein. The pH of the solution was necessarily kept near the neutral point to prevent hydrolysis of the sucrose used in the "bound water" determinations. If, as Bancroft and Rutzler (22) assume, coagulation is merely a physical aggregation of the colloidal particles of protein, which can be repeptized by addition of sugar, the explanation of the results ob- tained in the present work is evident. The results obtained by Bancroft and Rutzler do not account for the fact that heat coaguable proteins can be heated at pH valued above or below their isoelectric points with practically no flocculation. After cooling they flocculate readily at their isoelectric points without further heating. This indicates that some change, not admitted by Bancroft 35 and Rutzler, has taken place at the time of heating. The results of attempts to bring about a peptization of heat coagulated egg albumin presented in this thesis show practically negative results. Another possible explanation might be the chemical combination of the sugars with egg albumin thus preventing coagulation. The fact that egg albumin has been shown to contain an appreciable amount of sugar (40) (45) makes this possibility seem more probable. S. Frankel and C. Jellinek (41) successfully isolated a disaccharide from egg albumin which they found to be gluoosamino- mannose. Upon hydrolysis this compound yielded mannose and glucosamine. 0n the other hand, no record of the successful combining of unaltered sugars with protein is to be found. N. F. Goebel and I. L. Avery (42) found it possible to combine paraaminOphenol beta-glucoside with serum globulin. H. Pringsheim and M. Winter (43) claimed to have successfully combined glucose with the hydrolytic products of peptic digestion of proteins. A yield of 3 or 4% of the sugar-peptid product was obtained. Sdrensen, S. P. L. and Lorber, L. (44) critisize the results of Pringsheim and winter, claiming that their technique in sugar analysis would not give reliable results. None of the suggested possibilities for the mechanism of the protective effect of sugars against protein coagulation have sufficient experimental data to support them. It is evident that further investigation will be necessary before conclusions can be drawn. 36 37 SUMMARY It is possible to inhibit coagulation of egg albumin by heat or by ultraviolet light by addition of glucose, fructose, sucrose, mannose or mannitol. Egg albumin is completely stable to a temperature of 70°C when in a saturated solution of glucose or fructose. Egg albumin is only partially stable to a temperature of 70°C when in a saturated solution of mannitol. There is some indication that more water is "bound" by a solution of egg albumin in % glucose than in a solution containing no glucose. No flocculation occurs at the isoelectric point upon removal of the protecting sugar by dialysis after heating. This indicates that the denaturation reaction, not merely flocculation, has been inhibited. 2. 3. 4. 5. 6. 7. 8. 9. 10. BIBLIOGRAPHY Robertson, T. B., On the influence of temperature upon the solubility of casein in alkaline solutions, J. Biol. Chem., 5:147 (1908). Chick, H., and Martin, C. J., On heat coagulation of proteins, J. Physiol., 40:404 (1910) dichmann, A., Ueber die Krystallformen der Albumine, z. physiol. Chem., 27:575 (1899) Osborne, T. B., Vegetable Proteins, Second edition, Longman Green & Co., London and New York. Lepeschkin, w. H., The heat coagulation of proteins, Biochem. J. 16:678 (1922) Sdrensen, S. P. L., Proteins, Fleischmann Laboratories, (1924) wu, H., and flu, D.Y., Nature of heat denaturation of proteins, J. Biol. Chem., 64:369 (1925). Lewis, J. M. C., The crystallization, denaturation, and flocculation of proteins, with special reference to albumin and hemoglobin, Chem. Reviews, 8:91 (1931). Embden, G., Ueber den Nachweis von Cystein unter den Spaltungsprodukten der Eiweisskgrper, z. physiol. Chem., 32:94 (1901). M6rner, K. A. H., Zur Kenntniss der Bindung des Schwefels in den Proteinstoffen, Z. physiol. Chem. 34:207 (1901). 11. 12. 13. 14. 15. 17. 18. 19. 20. 21. 39 Johnson, T. B., Sulphur in proteins, J. Biol. Chem., 9:331 (1911). Arnold, B., Eine Farvenreaktion von Biweisskorpern mit NitrOprussidnatrium, Z. physiol. Chem., 70°3OO (1911). Osborne, T. B., and Guest, H. H., Hydrolysis of casein, J. Biol. Chem., 9:333 (1911). Harris, L. J., On existence of an unidentified sulphur grouping in the protein molecule, Proc. Roy. Soc., B 94:426 (1923). Young, B. C., The coagulation of protein by sunlight, Proc. Roy. Soc., B93:235 (1922). Rimington, C., Notes upon the sulphur linkage in wool, Biochem. J., 24:205 (1930). Corin and Ainsiaux, Bull. acad. roy. med. Belg., III. 21:355 (1891). Michaelis, L., and Rona, P., Die Koagulation des denaturierten Albumins als Funktion der dasserstaffionenkonsentration und der Salze, Biochem. 2., 27:38 (1910). Anson, n. L., and Mirsky, A. B., Protein coagulation and its reversal, J. Gen. Physiol., 13:133 (1929). Anson, M. L., and Mirksy, A. B., Protein coagulation and its reversal, 3. Gen. Physiol., 13:477 (1930). Anson, M. L. and Mirsky, A. B., The reversibility of protein coagulation, J. Phys. Chem, 35:185 (1931). 22. 23. 24. 25. 26. 27. 28. 29. 31. 32. 40 Bancroft, u. D., and Rutzler Jr., J. B., The denaturation of albumins, J. Phys. Chem. 35:144 (1931). Halliburton, a. D., The proteids of serum, J. Physicl., 5:152 (1884). Haycraft, J. B., and Duggan. C. w., The coagulation of egg and serum albumin, vitellin, and serum globulin, by heat, J. of Anat. and Physicl., 24:288 (1890). Hendrix, B. M. and Wilson, 7., A comparison of the titration curves of coagulated and uncoagulated egg albumin, J. Biol. Chem. 79:389 (1928). Lewis, P. 8. Kinetics of protein denaturation, Biochem. J., 20:979 (1926). Nu, H., Denaturation of proteins, Chinese J. Physiol. 3:1 (1929).‘ Booth, N., The denaturation of proteins, Biochem. J., 24:158 (1930). E Serensen, S. P. L. and Jargensen, B., Studies on proteins, Compt. rend. Lab. Carlsberg, 10:49 (1910). Chick, H., and.Martin, C. J., On heat coagulation of proteins, J. Physicl., 43:1 (1911). Chick, H. and Martin, C. J., On heat coagulation of proteins, J. Physiol. 45:61 (1912). 33. 35. 36. 37. 38. 39. 40. 41. 42. 41 Beilinnson, A., Stabalization of proteins to heat with sucrose and glycerol, Biochem. Z., 213:399 (1929). Teorell, Torsten, Protection of proteins in the presence of alcohol and acetate buffer, Biochem. Z., 230:1 (1950). Newton, R., and Brown, w. R., Frost precipitation of proteins of plant Juice, Canadian Journal of Research, 5:87 (1931). Spiegel-Adolf, Mona, Hitzeveranderungen des Albumins, Biochem. 2., 170:126 (1926). Gortner, R. A., Outlines of Biochemistry, John wiley and Sons, Inc., New York, (1929). Morrow, C. A., Biochemical Laboratory Methods, John wiley and Sons, Inc., New York, (1927). Allen, J. P., Accurate and adaptable micro-Kieldahl method of nitrogen determination, Ind. Eng. Chem. Anal. Ed., 3:239, (1931). Osborne, L. B. and Campbell, G. P., The proteids of egg white, J. Am. Chem. Soc. 22:422 (1900). Frankel, 3., and Jellinek, C., Uber die segenannte Kohlehydratgruppe im Biweiss, Buichem. Z. 185:392 (1927). Geobel, w. P., and Avery, O. L., Conjugated carbohydrate- proteins, J. EXP. Med. 50:521 (1929). .2 43. Pringsheim, H., and Winter, H., Die Zucker-Eiweiss- Kondensation, Ber. 6OA:278 (1927). 44. Sorensen, S. P. L., arn.Lorber, L., Die Zucker- Eiweiss-Kondensation, Ber., 60Az999 (1927). 45. Levene, P. A. and Mori, S., The carbohydrate group of ovomucoid, J. Biol. Chem., 84:49 (1929). INDEX Page I. Introduction II. Definition of Terms ....................... 1 III. Historical .0...OOOOOOOOOOOOOOOOOII0.000... 2 A. Whether protein denaturation is of a hydrolytic or de hydrating nature 0000000000000000000000000... 2 B. Liberation of sulphhydryl groups during denaturation ............... 6 C. Possibilities of reversing the de- naturation reaction ............... 9 D. The acid and base binding power of proteins before and after de- naturation OOCOOIOOOOOOOOOOOOOOOOOO 12 E. The possible liberation of ammonia or other nitrogenous compounds upon-denaturation ................. 14 "F. Factors influencing the de- naturation reaction ............... 15 IV. Experimental 000000000000000000000000000... 17 V. DichS810n 00.00.00.000...00000000000000... 32 VIC summary 000000000.0.0.00000.000000000000000 37 VII. 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