W 1 1 | \ W H I b ‘ ’l W Hill 1 I i \ "iN—x (/3010) SULPHYDRYL GROUPS AND THE COAGULATION OP PROTEINS AS ‘NFLUENCED BY REDUCING SUGARS The“: for thc Degree of Mn 3. WCHIGAN STATE COLLEGE Chester R. Hardt I94! gh. . .t . .. "v‘. a?“ I - .-l x- -J‘ fiNL!“A¢-“I'”-s -r‘!' . 4"... p— .\ .- r «N u. - '3 . ',; . cf“- fyran‘ ‘a‘ ‘ :94 l ”1 '9; A “ .1;"' .‘5‘ l...' ,. n '0.” a . 9- F9F'Igu x . m 9 t ‘;.>..P‘ vgoflw,‘ . , ‘ ‘IV SULFHYDRYL GROUPS AND THE COAGULATION 0F PROTEINS AS INFLUENCED BY'REDUCING SUGARS by Chester R. Hardt A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of EASTER OF SCIENCE Department of Chemistry 1941 1.138821 AC I’E‘IO 1-1'LE D GLEN T I wish to eXpress my gratitude for the helpful suggestions and straightforward criticism extended to me by Professor C. D. Ball. CONTENTS 1. Introduction 2. Historical ................................ A. The occurence of sulfhydryl groups in proteins ........... B. Methods for estimating sulfhydryl groups in proteins ........... C. Possible protein and carbohydrate combinations ................. 3. EXperimental .............................. 4. Discussion ................................ 5. Conclusions ............................... 60 Bibliography coo0.0000000000000000000000000 10 14 40 47 48 INTRODUCTION The study of proteins and the factors influencing their behavior has been greatly stimulated due to the grad- ual realization that a great many substances posessing re- markable physiological activities appear to be proteins. Certain enzymes, hormones, and viruses as well as toxins, antitoxins, antigens, and antibodies appear to be proteins. The terms denaturation, flocculation, and coagulation are confused in the literature on proteins. In this paper I will use these terms as outlined by Bull (1).. "Protein denaturation consists of an over-all process of three re- actions, and these reactions can under appropriate condi- tions, be separated and studied individually. The first reaction in the series is that of denaturation proper, which apparently is an intermolecular rearrangement whereby cer- tain chemical groups which were not detectable in the native protein are rendered so in the denatured product. Denature- tion is a necessary but not sufficient reaction in the pro- duction of coagulated egg albumin. The second reaction con- sists in the flocculation of the denatured molecules prepara- tory to coagulation. The flocculation reaction is the only one of the series that is reversible. The third and last step is the formation of an insoluble coagulum." The coagulation of proteins was one of theflfirst changes noted in connection with the stability of proteins. Coagu- lation changes the properties of proteins greatly and was regarded as a factor in life processes. In 1929 Beilinnson (2) reported that in the presence of sucrose, protein solu- tions were stabilized towards heat coagulation. Protection not nearly so great was reported for glycerol. Duddles (3) studied the effect of sugars and mannitol upon coagulation of egg albumin. He found that not only sucrose but glucose, mannose, fructose, and mannitol showed a definite inhibiting effect towards coagulation by heat and ultra-violet light. Newton and Brown (4) worked with plant saps and found that sucrose and glucose prevented coagulation of plant proteins by freezing. In a large number of cases when proteins denature sulf- hydryl groups appear (5). It is thus possible to use their detection as a measurement of denaturation. It is known that in a great many instances sulfhydryl groups are closely as- sociated with the physiological and chemical activities of proteins (26). In the present work I will attempt to show how certain factors influence the liberation of sulfhydryl groups as well as the estimation of these groups in native, partially coagu- lated, and completely coagulated egg albumin. HISTORICAL In reviewing the literature on this problem I will consider the following points. 1. The occurence of sulfhydryl groups in proteins. 2. Ksthods for estimating sulfhydryl groups in proteins. 3. Possible protein and carbohydrate combinations. THE OCCL-"i‘ibll-ICE OF SUIEHYDRYL GROUPS IN PROTEINS -In 1901 Embden (7) reported that he had isolated cy- steine from a protein hydrolasate. In the same year Morner (8) showed that this amino acid had been formed during the process of hydrolysis and separation and was in reality an artifact. Later Patten (9) was able to make it clear that cystine and not cysteine was the primary amino acid formed when proteins were hydrolyzed in the manner employed by Embden. Harris (10) cited work by Sieber and Schonbenko who obtained methyl and ethyl mercaptans by the fusion of proteins with alkali. He also refered to work by Rubner who obtained the same products by dry distillation and Drechsel who report- ed that during acid hydrolysis of certain proteins, mercaptans were formed and believed that they were derived from some basic body present in the original protein. Mgrner (8) first attempted to determine quantitatively the sulphur groups in proteins. He tested proteins for thio- lactic acid formerly isolated from horn protein by Friedmann, according to Harris (10), thioamino succinic acid believed by 2 Baumann and Schmitz (11) to be a protein constituent, cystine, and cysteine. From.the results obtained using egg albumin Mgrner concluded that cystine was the only one of these acids present in the native protein. Using a quantitative method based uponthe blackening of lead acetate paper Marner (8) reported that of the total sulphur present in ovalbumin only one third to one half of it could be accounted for by cystine. He noted also that in the case of ovalbumin an unidentified sulphur compound was volatilized and this represented about one third of the total sulphur. Suter (12) used a method similar to that used by'mgrner and reported analagous results on ovalbumin and on other proteins. Pick (13) working with primary protecses derived from fibrin noted that all of the sulphur was given off as hydrogen sulfide when treated with alkali. He interpreted this as indication that some of the sulphur present was in a form.other than cystine. Johnson and Burnham.(l4) suggested that this sulphur, other than cystine might be in the form of thiopoly- peptides and their derivatives. Johnson and Burnham.suggest- ed that oxygen in peptide linkages may be replaced by sulphur. Arnold (15) worked on a large variety of protein.material with the aid of the nitroprusside reaction. He found that tissue extracts, certain coagulated proteins, and native pro- teins gave a positive test. He believed that these positive tests were in all cases due to the presence of cysteine. Later Hopkins (16) was able to show that in tissue extracts the positive nitroprusside test was not due to free cysteine but to glutathione which he at that time believed was a di- peptide containing cysteine and glutamic acid. Using the nitrOprusside test of Arnold's with Hopkins modification, Harris (10), tested egg albumin in various states for cysteine. He found that raw egg white was non- reactive with nitroprusside but upon heat coagulation it gave a positive test. Furthermore he found that albumin precipitated by ammonium sulfate and weak alcohol gave a negative test but that albumin precipitated with hydrochloric acid gave a positive one. This showed that precipitation by hydrochloric acid was not simply a precipitation as had for- merly been believed. The white feathery precipitate resulting from ultra-violet light treatment gave a negative test with nitroprusside. The observation formerly made by Young (17) that this precipitate could be readily redissolved by shaking was confirmed. Somewhat different results were reported by Mirsky and Anson (5) who found that protein coagulated after irradiation with ultra-violet light gave a positive test for sulfhydryl groups and the quantity was the same as when the coagulum was further treated with strong acid. .Meldrum (19) stated that denatured proteins could be divided into three groups on the basis of their reaction with nitroprusside. 1. Those which gave a positive test for sulfhydryl groups Egg albumin was an example. 2. Those which gave a positive test for the disulfide group. Serum proteins were examples. 3. Those which did not give a reaction for either free sulf- hydryl or disulphide groups. Ovomucoid (10) and globin (19) examples. ‘ Mirsky and.Anson (18) did not believe that globin be- longed in group three because they obtained positive tests for both sulfhydryl and disulfide groups. Meldrum (19) re- peated this work and pointed out that the conclusions drawn by Mirsky and Anson were erroneous. However, Mirsky and Anson (20) in a later paper refutedeeldrum. . In a series of papers Mirsky and Anson (20) (21) (5) (22) brought out the following points. The number of sulf- hydryl and disulfide groups that can be detected in an un- hydrolyzed coagulated protein was equivalent to the total amount of cysteine and cystine that could be determined in the hydrolyzed protein. The appearance of sulfydryl and di- sulfide groups when proteins coagulated was the only known chemical change that occured in the protein molecule. Some native proteins contained detectable sulfhydryl and disulfide groups and the number measurable was always less than the number found when the protein was coagulated. The effect of denaturation was to shift the range of activity so that the sulfhydryl and disulfide groups could react at a lower pH. When coagulation was reversed thesulfhydryl and disulfide groups that became detectable upon coagulation were no long- er active, furthermore the behavior of these groups indicated that coagulation was reversible. The activity of sulfhydryl and disulfide groups was affected by coagulation, pH, temp perature, and the nature of the protein. Coagulation increas- ed the number of detectable sulfhydryl and disulfide groups. The effect that temperature had on the activity was not stated by the authors although they did say that it had a definite effect. Kirsky (25) pointed out that an increase in the activity of sulfhydryl groups without shifting the pH is the criterion of denaturation. Anson (24) believed that native egg albumin contained free sulfhydryl groups, but that these groups were relatively unreactive. By using iodine and iodoacetamide he was able to detect these groups even though they were unreactive towards ferricyanide, porphyrindine or nitroprusside. The fact that the properties of certain proteins were altered when placed in urea or other amide solutions, often with an increase in the number of detectable sulfhydryl groups was first reported by Hopkins (25). In a series of papers Greenstein and Coworkers (26) (27) (28) pointed out that when proteins are placed in solutions of urea, guanidine hydroch- loride, and related substances the number of detectable sulfhydryl groups varied widely. For all of the proteins investigated by Greenstein the maximum number of these sulf- hydryl groups was found in Solutions of guanidine hydroch- loride. The percentage of the sulfhydryl groups that could be detected was independent of the protein concentration and depended upon the concentration of the guanidine hydrochloride. Blumenthal and Clarke (29) stated that there was ample evidence in some proteins that sulphur existed in forms other than cystine, cysteine, and methionine. Ashley and Harrington (30) believed that part of the sulphur in zein was present as thiolhistidine and Zahnd and Clarke (51) have detected thioglyoxaline in fractions of hydrolyzed egg white. IMETHODS FOR ESTIMATION OF SULFHXDRYL GROUPS IN PROTEINS The earliest attempts to estimate quantitatively the sulfhydryl groups in proteins made use of the nitroprusside method described by Arnold (15). This reagent was consider- ed rather Specific for sulfhydryl groups and developed a pink color in the presence of these in alkaline solution. However, the color faded rather rapidly and several modifications have been suggested in an attempt to stabilize the color. Hopkins (16) used an excess of ammonium sulfate instead of aqueous mmonia. Walker (52) obtained better results by using sodium cyanide or potassium.cyanide. Zinc salts intensified and stabilized the color according to Giroud and Bulliard (55). Saturated solutions of sodium sulfate and magnesium.sulfate were used to accomplish the same purpose by mentzer (54). By varying the pH at which the protein and nitroprusside are allowed to react the reaction can be made more specific ac- cording to Zimmet and Perrenoud (55). Two methods and their applications for the determination of sulfhydryl groups were described by Mirsky and.Anson (5). The so called direct method in which the sulfhydryl groups are oxidized by cystine which is thereby reduced to cysteine. Consequently the number of sulfhydryl groups oxidized can be estimated by determining the amount of cysteine found. The authors stated that cystine oxidized all of the sulfhydryl groups and no other groups in the protein. In the indirect method oxidizing agents or iodoacetate were used to eliminate the sulfhydryl groups and the reagent added was then removed. The protein was then hydrolyzed and its total cysteine con- tent compared with the cysteine content of the untreated protein. The decrease in the cysteine content was equal to the number of sulfhydryl groups which reacted with the reagent. Cystine and cysteine in the hydrolasate were determined colori- metrically with phospho-lB-tungstic acid. Cysteine but not cystine gave a blue color in the absence of sulfite while cy- stine gave a blue color in the presence of sulfite. Kuhn and Desnuelle (56) introduced the use of the blue dye porphrindine for the estimation of reducing groups in- cluding sulfhydryl groups. This dye a powerful oxidizing agent reacts rapidly and stoichiometrically with sulfhydryl groups in the cold. Greenstein and co-workers (27) (6) (57) have used this reagent extensively for the determination of sulfhydryl groups in proteins. They noted that its applica- tion was based upon two assumptions. (1) That a positive nitroprusside test in a protein solution is given only by sulfhydryl groups and (2) that porphyrindine was reduced in a neutral protein solution at room.temperature within one to two minutes by sulfhydryl groups only. Greenstein also point- ed out that the carbonyl group gave a positive test but the tint of the color so developed was in marked contrast to the rapidly fading color given by sulfhydryl groups. Tyrosine formed an orange color that developed slowly and could be readily distinguished. Perez and Sandor (58) have reported satisfactory results using porphyrindine. Objections against the use of this reagent have been raised by Anson (59) on the grounds that it was hard to prepare, was unstable, and was a dangerously strong oxidizing agent for use on proteins. Todrick and Walker (40) reported a method which consisted in measuring the amount of the oxidation-reduction indicator phenolindo-2-6-dichlorophenol ( 2-6 dichlorobenzenoneindophenol) reduced by a known weight of protein. The indicator was first standardized in terms of cysteine hydrochloride. A series of test tubes containing the same amount of protein were prepared and varying quantities of the indicator were added. The con- tents were then allowed to react and the tubes that were de- colorized within twenty minutes were noted. A further series of tubes were then set up containing a closer range of quanti- ties of indicator. The authors claimed that under the condi- tions described by them the reaction was specific for sulfhy- dryl groups and accurate to two or three percent. Dickens (41) showed that under certain conditions of pH and temperature, halogenated acetic acids reacted readily with sulfhydryl groups forming the corresponding thio ethers and hydrogen halide. That iodoacetic acid can react with amino groups but slowly if at all under these conditions had been shown by Michaelis and Schubert (42). They pointed out that in the absence of an excess of iodoacetic acid the reaction between sulfhydryl groups and iodoacetic acid was much more rapid and the amino group was not affected. Smythe (45) has measured the rate of reaction of iodo- acetic acid with various sulfhydryl compounds by measuring the carbon dioxide liberated from.a carbon dioxide-~bicarbon- ate buffer as a result of the hydriodic acid formed. Rapkine (44) first showed that iodoacetic acid reacted not only with sulfhydryl groups of relatively simple molecules such as cysteine and glutathione but also with proteins. Based upon this observation was the method described by Rgsner (45) for the determination of sulfhydryl groups in proteins. In this method the hydriodic acid formed was treated with hydrogen peroxide and the color that developed was read on the photelometer which had previously been stan- dardized in terms of cysteine hydrochloride. hirsky and Anson (5) and Anson (22) found that ferri- cyanide in a neutral solution oxidized sulfhydryl groups to disulfide groups. They stated that cysteine was the only amino acid which was known to react stoichiometrically with dilute ferricyanide. These same authors concluded that con- centrated ferricyanide will oxidize tyrosine and tryptophane but these reactions were indefinite. To estimate sulfhydryl groups by this method ferricyanide was added to the protein solution, the sulfhydryl groups reduced the ferricyanide to ferrocyanide which was then estimated as prussian blue. These workers have obtained best results when the protein was dis- solved in Duponal P. C. solution. Flatow 47 has estimated the glutathione content of pro- tein solutions by using an excess of ferricyanide and back titrating with indigosulfonic acid. methods involving the use of iodine have been used by a number of workers; Okuda and Lasayoshi (4S), Tunnicliffe (49), Thompson and Voegthin (50), and Woodward and Fry (51). Iodine is believed to react with sulfhydryl groups forming disulfide groups and hydriodic acid. The hydroidic acid may then be titrated or oxidized and determined colorimetrically. This method has had rather wide applications but kirsky and lO Anson (5) believed it to be inaccurate for sulfhydryl groups at low concentrations. Anson (46) has stated that no reagent is available to determine without question all the sulfhydryl groups and no others in denatured egg albumin. POSSIBLE PROTEIN AND CARBOHYDRATE COHBINATIONS The retarding effect of carbohydrates on protein co- agulation may be due to peptization (52) or to a more definite chemical combination (55). According to Meyer (54) protein and carbohydrate combina- tions are divided into two main classes. The first, composed of the various mucopolysaccharides occur in nature either as free polysaccharides or as protein salts. The second group contains the glycoproteins; proteins or polypeptides contain- ing hexose amines and other sugars in an unknown combination. Chondroitinsulfuric acid a mucOpolysaccharide first isolated from cartilage by M3rner, according to Meyer (54), was noted by this author to co-precipitate with gelatin upon acidification. Meyer and co-workers (55) made use of this observation and prepared protein chondroitinsulfuric acid come plexes. After purifying these complexes by reprecipitation they were analyzed for hexosamine. The results indicated that the inorganic cation in the chondroitinsulfuric acid had been replaced by the basic amino group of the protein. These workers came to the conclusion that these combinations were of a salt like nature. Compounds of a similiar type were pre- pared using mucoitinsulfuric acid but they differed from the above in that they were more stable. ll Frankel and Jellinek (56) were able to separate a non- reducing carbohydrate fraction from egg white. From this fraction they isolated glucosamine and mannose. The same com- pounds were isolated from egg white and ovomucoid by Levene and Rothen (57) and Levene and Mori (58). These workers be- lieved that the non-—reducing carbohydrate was a glucosamine dimannoside. Frankel and Katchalsky (59) studied the reactions between mixtures of sugars and amino acids and polypeptides by follow- ing the change in pH occuring as the free amino groups disap- peared. They were able to detect a reaction when aldose sugars were used; a reaction depending entirely upon the alpha amino group of the amino acid. Reactions were detected over a pH range of 4.5-11.0 and an optimal pH for reaction was noted. In a later paper Frankel and Katchalsky (60) reported that glycine would combine with aldehydic sugars but would not cam- bine with non-aldehydic sugars. A definite crystalline acid was obtained by Genevois and Cayrol (61) by allowing a dilute cysteine solution of pH greater than 5.0 and formaldehyde to react mole for mole. Compounds were also formed using alanine and formaldehyde but the resulting acid was less stable. Slower reactions between cysteine and other aliphatic aldehydes were noted but reactions between cysteine and ketones were reported only when the ketones were present in large exceSs. The fact that thioglycolic acid and thiomalic acids did not react was explained by these work- ers as being due to the absence of an amino group in a beta 12 position to the sulfhydryl group. Schubert (62) by analysis of the crystalline compounds formed between cysteine and d-arabinose, d-xylose, d-glucose, d-mannose, d-galactose, and lactose showed that cysteine com- bined with these sugars in a mole to mole ratio with the eli- mination of a molecule of water. These compounds were formed at room temperature in a neutral solution and were rather easily soluble in water giving solutions that were acid to litmus. The fact that these compounds did not give positive nitroprusside tests under conditions where cysteine gave a strong test indicated that the combination was through the sulfhydryl group in cysteine. Schubert was not able to iso- late any compound from mixtures of fructose and cysteine indicating he believed that the linkage in the sugar is through the aldehyde group. Przylecki and Hajmin (65) (64) were able to form.comp1exes using proteins and various so called polyglucides. They found that below their isoelectric points, albumins formed salt like complexes with phOSphorylated glucides. Above their iso- electric point the complexes did not appear to be salt like. These workers presented evidence showing stoichiometric com- binations of myosin with various polyglucides. In a later paper Przylecki (55) stated that three kinds of combinations between carbohydrates and proteins were pos- sible, covalent, molecular and salt like. He also listed twelve ways in which carbohydrates might react with proteins through the free groups of the amino acids present in proteins. Przylecki was able to form compounds between maltose and oval- bumin, crystallized seralbumin, casein, and clupein. 13 In preparing these compounds which he called "symplexes", mixtures of the protein and carbohydrate werefiallcwed to stand at low temperatures for several days at a rather high pH. A parallelism between the lysine content of the protein and the amount of sugar that would combine with the protein was noted. No symplex was obtained using sucrose indicating that the reducible group of the sugar must be free for a re- action to take place. l4 EXPERIMENTAL The eXperimental work on this problem.was divided into three parts as outlined below. 1. The effect of sugars and mannitol on the liberation of sulfhydryl groups . 2. The effect of sugars on the coagulation of egg albumin. 2. An attempt to show protein--carbohydrate combination. THE EFFECT OF SUGARS AND MANNITOL ON THE LIBERATION OF SULFHYDRYL GROUPS Egg albumin was prepared by the method of Ketkwick and Cannan (65) in which sodium sulfate was used to precipitate the albumin. ‘A propeller type stirrer as suggested by Westfall (66) was used to dissolve the sodium sulfate and precipitate the egg albumin. The sodium sulfate was dialy- zed out of the solution under reduced pressure until the test for the sulfate ion was negative. The nitrogen content of the egg albumin solution was determined in duplicate by a,MicroéKjeldahl method (67). In this procedure 2 ml. ali- quots of the protein solution were pipetted into Micro- Kjeldahl flasks, 2 ml. of concentrated sulfuric acid, 0.1 gm. of potassium sulfate and a few crystals of copper sul- fate were added. The mixture was heated with a micro burner until it became light brown; a few drops of thirty percent hydrogen peroxide were then added and the mixture again heated. This treatment with.peroxide was repeated until the mixture was light blue. The distillation was then carried out in a Micro-Kieldahl distillation apparatus using boric 15 acid in the receiving flask. Titrations were carried out with 0.01 N hydrochloric acid. To study the effect of sugars on the liberation of sulf- hydryl groups the photelometer was standardized in terms of cysteine by the method of Rosner (45). Standard solutions of cysteine hydrochloride containing from 0.1 to 0.5 of a mg. of cysteine per 1.5 ml. were prepared. To these were added 4.5 ml. of distilled water, the mixture then heated at 70° C. for fifteen minutes and then allowed to cool. These mixtures were then treated with 2 ml. of potassium dihydrogen phosphate--sodium hydroxide buffer, pH 7.4,2 ml. of £25 N iodoacetic acid, previously neutralized with potassium hy- droxide, and then allowed to stand for thirty minutes. At the end of that time 0.25 ml. each of a solution ten percent in respect to both trichloracetic acid and concentrated sul- furic acid were added. This mixture was allowed to stand for five minutes and then filtered. To the filtrate was added 0.25 ml. of three percent hydrogen peroxide and the mixture made up to 10 ml. with distilled water. At the end of thirty- five minutes the color was read on a photelometer using a blue filter with a maximum absorption range of 4,500 A9--5,000 A9. The results of the standardization are shown in Figure 1. This standardization was repeated using sugar solutions in place of the distilled water giving identical results. An egg albumin solution was then adjusted to pH 4.8 with 0.1 N hydrochloric acid. One and one half ml. aliquots of this solution containing 6.5 mg. of soluble nitrogen per ml. were pipetted into test tubes and 4.5 ml. of the sugar solu- tion added. These mixtures were then treated exactly as in 33d 'S9N NI NOLLVELLNBDNOD ”1W Ol 8. H671 wz_ogroup is not necessary for inhibition but it may increase the amount. According to some workers in this field fifteen minutes heating at 700 0. causes one hundred percent denatura- tion and subsequent coagulation of egg albumin (40). If this be true the percentage of cysteine in denatured coagulated egg albumin as measured by the iodoacetic acid method is 0.590%. This value compares favorably with other values reported in the literature. The percentage cysteine in denatured egg albumin has been reported by various workers (26) (56) (40) to be 0.500%, 0.580%, and 0.650%. If 0.590% cysteine repre- sents the value for completely denatured egg albumin it is possible to calculate the amount of denaturation on the basis of the percentage cysteine detectable. On this basis since the percentage of cysteine calculated from the sulfhydryl groups was 0.561%, the percentage denaturation of the egg albumin in the presence of4¥ glucose would be:- .361 7555 x 100 . 51.15% 0n the same basis the amount of denaturation in the pre- sence of ¥.fructose where the average percentage of cysteine is 0.411% would be:- .411 .590 x 100 = 69.66% The percentage of cysteine in egg albumin denatured in the presence of;% mannitol is 0.481% and the amount of de- naturation would be:- jgga-x 100 81.52p The results obtained using the indicator method are in fair agreement with those obtained by the previous method. The percentage of cysteine in egg albumin dentured and co- agulated in the presence of glucose is in the latter case slightly greater than in the former one. Again it will be noted that glucose showed a definite inhibiting effect. Fructose and mannose also show inhibiting effects but the inhibition is less then when measured by the iodoacetic acid 42 method. By the indicator method the percentage cysteine in completely denatured and coagulated egg albumin is 0.570% assuming fifteen minutes heating causes complete denaturation. The percentage denaturation may, therefore, be calculated from the average percentages of cysteine found when egg albumin is heated in the presence of l}: glucose and if: fructose where the average values for cysteine in the presence of these sugars were respectively 0.578% and 0.455%. The percentages denatur- ation would be :- 0378 = J.’ 75-7-6 X 100 66.13/0 and 4%?)- x 100 = 79.82% The percentages denaturation in these cases are slightly greater and the percentage protection therefore slightly less than in the cases of the same sugars measured by the iodoacetic acid method. Because of the inability to differentiate between small amounts of the indicator this method was not considered very reliable. The inhibiting effect shown by the pentoses, 1-arabinose and d-xylose, can again be used to calculate the percentage denaturation. The average percentage cysteine found in the presence of 4:1 arabinose was 0.406% by the iodoacetic acid method and the percentage denaturation would be:- .406 - 755-5 1: 100 - 68.81% In the case ofE xylose the percentage cysteine found I-' averaged 0.455% and the calculated percentage denaturation would be:- 43 .590 x 100 75.38% The inhibiting effect of the pentoses is slightly less than the inhibiting effect of the hexoses when measured by the same method. The pentoses l-arabinose and d-xylose show a definite inhibiting effect against coagulation measured by the resid- ual nitrogen method as shown in Tables 10 and 11. Arabinose showed a greater amount of inhibition than xylose but both showed definite inhibiting effects that were proportional to the concentration. To bring a substance into colloidal solution is called peptisation. Lloyd and Shore state that, Waworse term than peptmation, with its suggestion of peptic digestion, would have been hard to find." (70) The assumption of Bancroft and Rutzler (52) that protein coagulation is merely a physical aggregation of the colloidal particles of protein, which can be repeptised by addition of sugar was not entirely verified in the present work. According to Bull (1) before a protein can be coagulated it must be denatured and the denaturation process apparently causes certain changes in the protein molecule such as the liberation of sulfhydryl groups. Bancroft and Rutzler do not consider the denaturation process in their explanation of the prevention of coagulation by sugars. This present work shows that both denaturation and coagulation are inhibited by sugars and mannitol. If this inhibiting effect is due to combinations of a more stoichiometric nature further eXperiments of the type Pryzlecki carried out might have offered an eXplamation (55). 44 Under certain conditions of time, temperature, and pH Pryzlecki has been able to obtain "symplexes" of a stoi- chiometric nature between proteinsfiand carbohydrates. The conditions under which the influence of sugars on the coagu- 1ation of egg albumin were studied as previously described in this present work would not favor the formation of symplexes according to Pryzlecki. The same would be true of the work carried out by Buddies (5). If the formation of symplexes does inhibit coagulation it should have been possible to demonstrate the effect by running simultaneous experiments under the conditions used by Pryzlecki and also as previously described in this work. The percentage nitrogen remaining in the filtrate from.samp1es of egg albumin coagulated at pH 4.6 and at pH 8.6 as shown in Table 12 make it clear that a high pH causes less coagu- lation of egg albumin. Since the isoelectric point of egg albumin is approximately 4.8 a coagulum readily appear at this pH while at pH 8.6 a precipitate forms but it does not form a typical coagulum. If this mixture is then adjusted to pH 4.8 by the addition of a suitable buffer a coagulum appears. The percentage nitrogen in the filtrate from.the latter case is much greater than in the former. This indi- cates that not only coagulation but denaturation as well is inhibitedat higher pH values an observation fully described by Sorensen (69). Table 12 shows the inhibiting effect of glucose at both high and low pH. The amount of inhibition can be calculated at both pH values from the average per- centage nitrogen in the filtrate. 45 The average percentage nitrogen in the filtrates coagulated at pH 4.8 is 8.42% but in the presence of .5 M glucose at pH 4.8 the average percentage nitrogen in the filtrate is 12.66% therefore the percentage inhibition would be:- 12066 - 80423 8.42 x 100 = 51.50% when egg albumin was coagulated in the presence of .5 M glucose at pH 8.6 the average percentage nitrogen remaining in the filtrate was 58.58% therefore the percentage inhibition would be:- 58.58 - 48.20 48.20 x 100 = 21.10% It appears that glucose shows a greater inhibiting effect at the lower pH. The influence of fructose can be calculated in the same manner referring to Table 14. At pH 4.8 the aver— age percentage nitrogen remaining in the filtrate when albumin is coagulated in the presence of .5 M fructose at pH 4.8 is 12.52% and the percentage inhibition would be:- 2.52 - 8.42 x 00 = 48.6 4 8.42 l 0” At pH 8.6 the average percentage nitrogen remained in the filtrate was 45.96% which is less then the average value found for the albumin alone at this pH from these results it appears that fructose does not show an inhibiting effect at the higher pH. Other workers (60) (61) and (62) have reported that non-aldehydic sugars would not combine with proteins, amino acids or polypeptides and this may have some connection with this present work. However, Pryzlecki (55) believes that "symplexes" are formed when the sugar contains a reducible gr 0111) e 46 The influence that d-glucose and d—fructose have,under the conditions specified by Pyrzlecki as shown in Tables 15 and l6,seems to indicate that standing for considerable periods of time causes no greater inhibition. Since standing under these conditions should permit the formation of "symp p1exes"according to Pryzlecki it would seem that the f0rmation of these ”symplexes' does not increase the inhibition of pro- tein coagulation by these sugars. This present work does not indicate therefore, that the formation of these so called "symplexes‘ is the mechanism of the prevention of coagulation. 2 The results obtained in the attempt to show a combination between egg albumin and sugars as shown in Table 17 indicate that the sugar and the protein do not combine. The average result for the reducing substances found when egg albumin was coagulated in the absence of glucose is slightly higher than the average result obtained when egg albumin was coagulated in the absence of glucose. The results being 1.501 mgs. in the presence of glucose and 1.268 mgs. in the absence of glucose or an increase of 2.60% in the presence of glucose. However, variations as great as will be noted in individual successive trials and the difference in probably not signifi- cant. Since the coagulum in the above work was washed with distilled water in an attempt to remove any absorbed glucose it should not be concluded that no combination occured. An unknown labile combination of a very readily hydrohsable type that could readily be decompSed by washing with distill- ed water may have resulted. This experiment under these con- ditions did not show such a combination. 4. 6. 47 CONCLUSION Denaturation of egg albumin by heat was inhibited by the presence of d-glucose, d-fructose, d-mannose, mannitol, l-arabinose and d-xylose. The amount of inhibition was froughly proportional to the amount of sugar. This denaturation can be measured by the determination of sulfhydryl groups both by the iodoacetic acid method and less satisfactorily by the use of 2-6 dichlorobenze- noneindophenol. 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