THE EFFECT OF SUGARS ON SOME PHYSECAL AND CHEMECAL PROPERTIES OF EGG ALBUMQN Thais for H‘IO Dogma of Ph. D. WCHIGAN STATE UNiVERSlTY Edwin Loo Baker 1956 LIBRARY / , ( ‘ PRCPF‘T‘TTY or I’ ME'br‘ELEI'U'J DU”; UH‘ILHS‘TY PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE NOV 2 1 2005 A 6/01 c:/CIFIC/DaIeDue.p65-p. 15 TILE EFFECT OF SUGIIRS ON SCH-7F. PHYSICAL AI‘CD CHE-TICAL PROPERTIES OF EGG ALBUI—EIN Edwin Leo Baker A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture end Applied Science in pertiel mums of the requiremente for the degree of DOSTOH CF PTIIWOPHI Eepartment of Chemistry 1956 AC mmmomrrs I wish to empreee aw deep appreciation to Proteeeer C. D. Hall for hie invaluable guidance end eneouregement during the course of thie work. I else wish to expreee Iv einoere gratitude to Dr. Bane Lillevik for hie helpml meantime end ueietenee in the second part of thie work. i1 M It previoml: we chem that eeveral eimple sugars, one corre- epcnding alcohol and e dimcheride inhibit the precipitation of egg manna brought ebout by heat et We. end 138 I..8. Since no more protein precipitated when the sugar wee dielyzed out o! the mixture theee experimente were interpreted ce meaning that the denatureticn of egg albumin wee inhibited rather than the precipitetion ct otheruiee denetured meterial. Retire egg elbtmin doee not react with typical eulfhydryl re- egente uhereee denetured egg elbundn reacte with euch reagente in e quentitetive manner. It previously wee ehovn that eugere end related empounde else inhibit the liberetion of mlnwdryl groupe in egg elbtmin brought ebout by heat. Since ensure do not interfere in the reaction of free eulfiwdryl groupe with euch reagente theee eXperimonte were taker. on further proof that eugnre end releted compounde protect egg albumin againet the denaturing effecte of heet. The neeeuremente of mlfhydryl gum in the uperimente above indiceted e cysteine content of 0.59 per cent in denatured and pre- cipiteted egg elbumin. Leter cathode of meuring mlflzydryl grcupe heve indicated e cysteine content of 1.2 per cent in denatured egg elbmin which we kept in eclution during the measurement. Adapting these letter nethede the present work hee ehown thet 1.0 n gluceee, gelecteee, tructeee er eucroee prevent about 30 per cent of the eulflvdryl groupe ueually found in egg albumin heated at 50°C. and pH 1.0 Iran appearing. Thie ie mrther proof that augere protect egg albumin against acid-heat denaturation. hhen guanidine hydrochloride or sodium dodecyl euli'ate ia ueed to denature and keep egg albumin in eolution for eulfmrdryl meaeure- meat, the sugars glucose, fructoee. galactoee or eucroee do not affect the amount of euli’hydxyl groupe usually measured. Thie ie evidence that eugare do not affect the denaturation of egg albumin brought about by either of those magenta. Since charged groups in the protein molecule are usually thought to be important factors in denaturation, it seemed poeeible that the sugars covered up some critical charged groups and thue affected egg albumin denaturation. Such a circumstance could be indicated electmphoretically. Electrophoreeie eXperincnte did not demonstrate any combination between egg albumin and glucose, fructose, eucroee, or mannitol detectable as a new component. This indicates that the eugere do not in any way affect the charged groups in egg albucdn in preventing denaturation. Egg albumin heated at 50°C. and pH 3.0 showed two componente when analyzed electrophoretically at pH 3.0 in glycine-hydrochloric acid buffer. (he of thoee components: was precipitated at pH l..8 and thue wae indicated to be denatured egg albumin while the other component wee eoluble at pH [“8 and thus was indicated to be native iv egg albumin. The precipitable component was shown to foretelouly when egg albumin was heated in the presence of 1.0 H glucose, fructose .nannitol or glycine. Approximately the same amount of denaturation occurred in twenty-four hours in the presence of glucose or eannitol ee occurred in twenty minutes in egg albumin alone when heated at so‘c. and pH 3.0. Electrophoresie was thus shown to be another means of indicating that sugars and related compounds protect egg albumin against denaturation by heat. TABLE OF CONTENTS Page Iz;mmmn(m00000000000000.9.3.0...000......O0.00.0.0...0.0.00... ml'ysn'13nu - PAP-T 013.000.00.000.0...OOOOIOCIIOQOOOOOOCOO00.0.. Hateriale..................................................... Detection Of Denaturaticn..................................... Denaturing Agents............................................. RESULTS - PART mEOOCOOOOOO0.00.00.00.00...C0.0.0.0.0000...00.... Denatureticn by Guanidine HFUIOCHIOridOeeeeeeeeeeeeeeeeeeeeeee ”0393.1111th by Sodium Eodecyl Sulfate........................ DCDIthItiOfl.hy’HOCteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee DISCUSSICN4. PART ONEeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee EXPERR'RIITAL - PART WOOD...OOOOOOOOOOOIOOOOOOOOOOOOOOIOOOOOOO... Materillleeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Solutions fbr’ElOOthphOPOBIIeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee E106tf0ph0f081l Apparatus..................................... RESULIS O’PART TWOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Electrophoresis It pH Seleeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Electrophoresis ‘3 DH 6e8eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Electrophoresis O‘ pH 3e0eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee DISCUSSICNIO PART TWOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee COT‘CLUSIwSOOIOOOOOOOOOOOOOOOOOOOOOOO..OOOOCOOOOOOOOOOOOOOOOOO0.. umm CWOOCOCOOOOOOOOOOOOOOOOOOOOGOOCOOOOOOCOOOO000...... fifigfifitfifitfigfisqu 0‘ \fl \flkfi ta \8 ‘4 )3l9h) A) E Figure l. 2. 3. 1.. 5. 6. 7. 8. 9. 11. LIST OF FIGURES Page Electrophoretic Patterns of Native Egg Albumin, Egg Albumin with GIUOOBI and Glucose Ohlykeeeeeeeeeeeeeeeeeeee 33 Electrophoretie Patterns of native Egg Albumin, Egg Albumin with Glucose, Egg Albumin Heated with Glucose..... 35 mectrophoretie Patterns of Egg Albumin Heated at 50°C. for Six Hours in the Presence of Glucose and Sucrose at Different pH VEIUOIeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 36 ElectrOphoretic Patterns of Native Egg Albumin, Egg Albumin Heated at 50 ‘C. for Wenty Hinutee, Alone and filth GlUCOIOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 38 Electrophoretie Patterns of Egg Albumin Heated at 50‘C. for‘Verioue Times in 2.0 M GIHOOBOeeeeeeeeeeeeeeeeeeeeeeee 39 Electrophoretic Patterns of Egg Albumin Heated for Trusty- tour Hours at 50'C., pH 3.0, in 2.0 H Glucose, and of the Supernatant and Precipitate Resulting Upon AdJustment to pH “OBCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. w flectrophoretic Patterns of Native Egg Albumin, and Egg Albumin Heated for 20 Minutes Ct 50.Ce. pH 3e0eeeeeeeeeeee 53 Electrophoretic Patterns of the Precipitate and of the Supernatant After Adjustment to pH l..8 of Egg Albumin, Heated for 20 Minutes Ct 50°Ce’ pH 3e0eeeeeeeeeeeeeeeeeeee hh Electrophoretic Patterns 01‘ Egg Albumin heated for Six Hours at 50 0., pH 3.0, in 2.0 H Glucose, and of the Super- natant and Precipitate Resulting Upon Adjustment to pH l..8 1.5 Electrophoretic Patterns of hgg Albumin Heated for Six Hours at 50 0., pH 3.0, in 2.0 M Glucose, and of the Supernatant and Precipitate Resulting Upon Adjustment to p“ ‘08.00.0.00...OOOOOOOOOOOOOO0.0000000000000000000...... “6 Electrophoretic Patterns 01' Egg Albumin Heated Alone for 6 and 2h hours, 8% SUOCe. ph BeOWeeeeeeeeeeeeeeeeeeeeeeeee so LIST CF FIGURES - Continued 12. 13. 11.. 15. 16. Electrophoretie Patterns of Egg Albumin Heated for Six and Mnty—tom' Hours at 50°C., pH 3.0, in the Presence 0: 2.0 H GluflOOIeeeeeeseeeeeeeeeeeeseeeeeeseeeseeeeeeeeee mectrophoretic Patterns of Egg Albumin Heated for Six and Tuentyu-four Hours at 50°C., pH 3.0, in the Preseme 0f 2e0 H Hannitol........................................ mectrophoretie Patterns of Egg Albumin Heated for Six and Twenty-four Hours st SO'C., pH 3.0, in the Presence 0: 2.0 H FM“”OOOOOOOOOOOOOOOOOOOOOOOOOOOOOCO0.0.0.... Electrophoretic Patterns of Egg Albumin Heated for Six and Mtybfour Hours at 50‘0., pH 3.0, in the Presence or 2.0 M GlyCiHOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseseeeeeeeee mectrophoretio Patterns of Egg Albumin Heated for Six and Twenty-four Hours at 50’C., pH 3.0, in the Presence Of 2.0 H GlchrOleeeeeeeeeeeeeeeeeeeeeeeeseeseeeeeeeeeeee Page 51 55 Table I. II. III. LIST OF TABLES The Hobility of the Components Show in Figures 1.,5 and 6.. Th. Mobility 0: th. Cunponents Shown in Figures 7‘10eeeeeee The Mobility of the Components Show: in Figures 11-16.”... Page 1.1 L7 56 INTfiODUC TI ON HITRODUC TI CE Neuberg (l) in 1916 described the characteristic ability of water solutions of numerous salts to dissolve otherwise insoluble materials. He called this property hydrotmpic and pointed out that a wide range of materials including the proteins were affected by it. He described in particular several experiments with sheep blood serum and egg yolk in which some hydrotrOpic salts, such as sodium bensoate and sodium salicylate, maintained a clear solution even when the mixture was heated. Later, following the lead of colleagues who had shown that 4 several anticoagulants protected the biochemical properties of diptheria toxin and antitoxin against heat, Beilinsson (2) proposed to determine whether all the physical-chemical preperties of typical proteins were preserved by the presence of such hydrotropic materials. He worked with two protein preparations, 1) rabbit serum and 2) five per cent egg albumin in ptwsiological saline, and two anticoagulants, 1) glycerol and 2) sucrose. The method of analysis was titration of aliquot samples with saturated ammonium sulfate solution to a standard turbidity. By plotting the milliliters of saturated amonium sulfate required against time of heating, Beilinsson was able to show the following: 1) The stability of the protein materials decreased as the temperature increased. 2) The stability increased as the concentration of the stabilising agent increased. 3) Sucrose was a considerably better stabilizing agent than glycerol. In fact, rabbit serum saturated with sucrose was almost completely stable for one hour at 62°C. and pH 7.1, and egg albumin saturated with sucrose was stable for one hour at 75°C. and pH 7.1. Experiments, in which the quantity of nitrogen in the protein precipitated at the isoelectric point (pH 5.3) was determined, confirmed the results given above. Duddles (3) in this laboratory expanded the work of Beilinsson by using several hexose sugars and one corresponding alcohol as well as sucrose. Denaturation and subsequent coagulation of the protein, egg albumin, was accomplished by heating at 70°C. for ten minutes at pH l..8. The quantity of nitrogen in an aliquot of the filtrate was then determined. The results showed the following: 1) In all cases the mat of nitrogen in the filtrate increases as the concentration of the stabilizing agent increased. 2) Glucose was the best stabilizing agent followed closely by fructose; egg albumin saturated with either glucose or fructose was almost completely stable to heat at 70‘C. for ten minutes at pH 5.8; mannitol was fairly effective and narmose was slightly effective. 3) Glucose stabilised egg albumin to coagulation caused by ultra-violet light at pH l..8. I.) Coagulated egg albumin was not repeptiaed by satura- tion with glucose and subsequent standing for twenty-dour hours. The work of Beilinsson and Duddles merely proved that sugars and sugar alcohols inhibited the heat coagulation of egg albumin; proof that denaturation was prevented'was lacking. Hardt (h) approached the problem.by using;the fact, first demonstrated by Arnold (S), that heat denatured egg albumin does react with sulfhydryl reagents whereas native egg albumin does not. Two methods were used to measure sulfhydryl groups: 1) The method of Rosner (6) using iodcacetate and 2) The method of Todrick and Walker (7) using phenolindo-2-6—dichlorophenol. The first method seemed more reliable but the second method confirmed the results of the first. Denatu- ration was caused by heat st 70°C. for fifteen minutes at pH l..8. The following observations were made: 1) The number of sulfhydryl groups liberated decreased as the concentration of sugar, ranging from 0.01.5 H to 0.1.50 M, increased. 2) Glucose was the best sta- bilizing agent with fructose and mennosc following; mannitol was not nearly so effective; archinose was as effective as fructose or mannoss but xylose was slightly less effective. 3) Both arsbinose and gloss stabilized egg albumin when a method of analysis similar to the one employed by Duddles was used, but xylose was less effective. A) Egg albumin was more stable at pH 8.6 than at prh.8. 5) The effectiveness of glucose or fructose in.stsbili- sing egg albumin was not improved by eXposing the protein to contact with the sugar for*periods up to ninetybeix hours before denature- tion'by heat. 6) No proteinpcsrbohydrate combination could be demonstrated. The work of Beilinsson, Duddles, and hardt had demonstrated by three very different cathode of analysis that several. sugars inhibit to a greater or lesser degree the denaturation and subsequent coagu- lation of egg albumin. Yet the nature of this inhibition had not been eXplained. [Islet had found only 0.590 per cent cysteine in denatured coagulated egg albumin using the icdoacetats method. This value agreed quite well, however, with the results of several other workers (1.) including those by kinky and Anson (8) who gave values of 0.56 per cent and 0.616 per cent cysteine for egg albmnin with two different respective methods. Later, however, Anson (9) showed that 1.2 per cent cysteine could be measured in donatired egg albumin dispersed by the denaturing agent. he achieved this result with very different titrating agents, each in the presence of several different denaturing agents, all of which kept the denatured protein in solution. "The fact that very varied procedures yield the same titration value is strong evidence that only wlfhydryl groups are being titrated.“ (10). Since twice as many sulnwdzyl groups were revealed by these methods as by the methods used by Hardt with coagulated egg abut-Lin, Anson (11) had suggested that the inhibiting action of sugars should be determined by case of the latter methods to make sure of the validity of the previous work. It was with this suggestion in mind that the first portion of the experimental work was undertaken. Luck and co—workers (12, 13, 15) performed a considerable amount of work with the heat stabilization of human scram alburin and bovine some albumin during ':--.'orld Liar II. They worked mostly with 25 gram per cent protein solutions because that was the form in which the human serum albumin was nmfac'tured and distributed for use by the Armed Forces. A rapid micro-method of analysis was develOped in vmich the solutions were heated in thin-dralled capillary tubes and the “cloud point” time observed (12). They found that the basic, requisite structure for stabilizing properties was an anion with s non-polar group. The madman stabilisation was attained with salts of fatty acids 7 to 8 carbon atoms in chain length and in s concentration of about 0.15 B. Cations with the same non-polar groups increased the susceptibility of serum albumin to heat denatu- ration. It was shown that these effects were the same on both sides of the isoelectric point of albumin. The strength of the acid group did not markedly effect the mount of stabilization since the relative effect of the sulfonic and carboxy acids with alkyl amps of the same chain length was about the same. Viscometric studies showed that the fatty acids prevented viscosity increases in heated solutions of albumin thus demonstrating that the fatty acids stabi- lised native and not denatured protein. Ultrafiltrstion studies permitted the determination of the amount of bound fatty acid anion in a solution containing both semm albumin and the sodium salts of lower fatty acids. It was found that the mmnt of combination of fatty acid anions with serum albumin increased markedly with an increase in the chain length of the fatty acid. The amount of combination of caprylate was decreased considerably'if the albumin was denatured by urea prior to the addition of the caprylate. Electrophoretic studies (16) demonstrated e specific interaction of the fatty'ncid anions with albumin with an increasing affinity in escending from.butyrete to capryiste. Lundgren g; Q. (17) had demonstrated the formation of complexes between egg albumin and detergents of the elkylaryl- sulfonste type in solutions alkaline to the isoelectrio region. These complexes exhibited well-defined electrophoretis boundaries whose mobilities ranged between those of the protein end detergent. Putnam end fieursth (18) were able to show two complexes between horse serum albumin and purified sodium dodecyl sulfate which had wellpdefined electrOphoretic boundaries. The formation of these complexes was dependent upon the weight ratio of protein to detergent rather than on their absolute concentrations. This finding had been confirmed by precipitation and viscosity studies on horse serum albumin and sodium dodocyl sulfate mixtures. Although no complexes of protein and non-ionic materials had been.demonstrsted previously it was this work of Putnam.and heursth which prompted the second portion of the present work, that is, an attempt to demonstrate complex.fornntion between egg albumin and various sugars by means of the electrophoresis apparatus. HPERB‘E‘JTAL - PART (NE 331311112 ii’IETfi .- Fidl'l‘ GEE native e353 slbmgin neither gives a detectable nitropmsside color for suli'hydrfl groups nor will it react with such mid oddizing agents as porpm'rindin, ferricyanide ion, and tetrsthionste ion. when e33 slim-tin is demtured by heat or by a mtber of other physical and classical scents, it readily gives a nitrOprusside color test for sulfhydrjl groups and will react with several reagents in e quantitative summer. Consequently the measurement of mzlfllydryl groups can be used to detenine the mmxt of densturation which has tel-zen place in a given sszotmt of ex albxrain. As mentioned previously, Anson (l9) hss developed several nethods of measuring sulflvdryl g roups which indicate tries the umber of sulfilydmrl groups that previous methods have sham. It was the purpose of the first part of the commented work reported in this dissertation to adopt the methods of Anson so that the effect of sugars on the mount of dmnturstion of egg clan could be observed better. IZ’L'E" 17111113 All inorganic chemicals met either 0.1”. or A.C .3. specifications and were products of either the J .T. Baker Chemical Caspsrw or Baker and Adamson. The sugars used were C.P. products of the Pfanstiehl Chancel Company. The simple sugars were the natural isomrs. Several samples of egg alburin were prepared by the method of Keksick and Cannon (20). All were recrystallized three times and then redissolved in s ninirmm amount of water. Part of some preparations was kept for extended periods of time under toluene in the refrigerator. All solutions were diolyzed against distilled water to remove sodium sulfate (which use occluded with the crystal- line egg albumin) before the solution was used in experiments. Dialysis was done both at room temperature and in the refrigerator but in all cases was continued until the specific conductance of the albumin solution was less than 2 x 10" aha. After dialysis, solutions were kept under toluene in the refrigerator. The final cementrstion was determined by nitrogen analysis (21) or (22). The methods developed by Anson (19) utilising potassium terrin- cyanide were the simplest to perform; the reagent was readily obtainable and stable in solution. Other procedures developed by Anson with p-chloro-memuribmsoste and tetrathionste ions were used by his only to confirm the results which he obtained with potassium ferricyanide. In several procedures using the latter reagent the results were obtained by developing Prussian blue from the ferro- cyanide previously famed when ferrlcyanide reacted with the sulf- hydryl groups of denatured egg albtmin. The amount of Prussian blue was determined by taking a reading in a suitably calibrated photelo- meter.‘Ir It was necessary, therefore, to have s solution of potassium fen'ocyanide which could be used as a reference standard. * A photoelectric calorimeter. Three procedures were used for making potassium ferrocymide stock solutions during the course of this work. 1) me potassium terrocyanide contained three moles of water of crystallization and had a molecular weight of 1.22.33. 'mue, 1.0558 3. made to 250 :21. resulted in a 0.01 M solution. 2) the same lot of potassium ferrocynnide was beams to constant weight at ios-no‘c. It was then assumed to have no water of crystallization and a molecular weight of 368.272. than 1.8412 g. was 218.60 to 100 m1. at 0.05 E solution resulted. Sodium carbmste in 1 concentration or 0.2 per cent was added to this solution as e stabilizer. 3) For the greater part of this work the mount of potassium ferrocyanide in accurately weighed samples was determined by titration with standard potassium permanganate solution (23). From this titration the material was calculated to contain 101.6 per cent KhFe(CI£)6 - 3 820, indicating a partial detvdration. Therefore subsequent stock potassium terro- cyanide solutions were made of 1.0391. go of this suit diluted to 250 ml. to give a 0.0115 solution. ‘I'I'Iese solutions were stabilized by 0.2 per cent sodium carbonate. Fresh potassium ferrocyanide stock solutions were usually made monthly; all solutions were diluted to 0.001 M for use as reference standards. Potassium ferricyenide solution was rude according to the directions of Anson (19). The stock solution was 0.1. if; and any ferrocyunide present was oxidized by treatment with brosdne. The terricymide solution we stored in the refrigerator and usually 10 1.25 ml. of the solution was diluted to 10 ml. to mks a 0.05 2*: solution for use. The stock solution was tested occasionally for ferrocyanide with ferric sulfate and subsequent develcpacnt of Prussian blue. Ferric sulfate solution, used to develop Frussisn blue from ferrocyenide, me made according to Folin end thlmroe (25). Sulfuric acid in e concentration of 1.0 K was made and standard- ized. It was used to stop the oxidising notion of potassium ferri- cyanide when the mat of sulfhydryl groups was to be detcrndned by the development of Prussian blue. Two buffers were used during the course of this work: 1) e neutral hitter, pH 6.7-6.8, consisting of equal molar ports of 1 H NaHZPO‘ end 1 14 “2mg, end 2) e buffer, pH 6.3, consisting of one part 1 l! RagllPO‘ and three parts 1 H HafizPOh. The pH of each of these buffers was measured on the Beckmn ph motor. In the procedures developed by Anson (1?) involving the develop- mom. of Frussiun blue the usual reagents were as follows: 1) 0.5 all. of two per cent egg albmnin or 1.0 ml. or one per cent egg abusing 2) neutral phosphate buffer varying in mount from two drops to 0.2 ml. depending on the procedure; 3) ferricymide solution which varied from one drop of 0.5 M solution to 0.5 ml. of 0.1 H solution; I.) end the demturing agent mic}: could be urea or sodium dodccyl sulfate. These reagents were allowed to stand together at 37 °C. for e mum. length or time varying has one minute for sodium dodecyl sulfate to rm minutes for um. This period of time was to poms. the reaction between denatured egg, albumin end ferricyanide to occur at e neutral p31. '31. reaction was stopped by the addition of 0.5 ml. or 2 H sulfuric acid, then water was added to e voltme of 9.5 nl.. and finally 0.5 ml. of ferric sulfate was added to develop Prussian blue. After tmty nimtes the mount of Prussian blue was deter— mined by use of e"p§wtelomter’ueing light transmitted by e red filter. The liellige-tiller ”photelozneter'eet at 660 an m the instrwzmt used in the present work for measuring the concentration of Prussian blue. As indicated above the usual amount of egg nlbmin was 10 mg. which, upon denaturstion, reacted with terricyanide to tone one :11. of 0.001 M ferrocymide. aerei‘ore, mints of terrocyanide from one 821. to 0.0 ml. were used with e final volme of reagents to ten ml. to proper-e e standard curve. The procedure was as follows: To moored qumtitiee of 0.001 2% potassium ferrocysnide were added 0.2 ml. of buffer, pi! 6.8 or pH 6.3; 0.5 ml. or ten per cent sodium dodecyl sulfate; from one drop to 1.0 E1. of 0.05 I"; ferricysnidex water to nine 12-1. and finally 0.5 ml. of 2 fl sulfuric acid and 0.5 ml. of ferric sulfate. After twenty nimtes the per cent trauma-is- sion was LTOBSUM in the Eiellige—f'iller"photelomter' at 660 sub Such curves were run seversl times during the course of the work always with the same results. The presence of egg albumin did not change the values when the experiment wee errenged so that the denatured material and ferricyanide could not react. no attempt was node to keep the ionic strength constant in the experiments reported in this part of the dissertation. Several reagents, i.e. buffer, detergent, and potassium terricysnide, which had an effect on the ionic strength were present 3 the variability in the newts of these reagents present from experiment to experiment made the maintenance of e constant ionic strength ineXpedient. Furthermore, Luck end coworkers (13) bed found thst ionic strength was not an importent factor in their work with heat densturetion of serun elbumin. Such en usunption my be questionable. fliATURING AGEtlTS 1. Uree. Ures which use the denaturing egent in some preliminsry experi- Isnte use used es obteined. A few experiments were carried out using uree es the denaturing egent but these experiments were discontinued when it was discovered thst egg elbunin precipitated at the time or the Prussian blue development. Further, the lsrge quantities of urea needed to bring shout densturetion made it en inconvenient experimental procedure. l3 2. Gumidine ib'drochloridc. Experiments with guanidinc hydrochloride used forricyanido as a titrating agent and the nitroprussidc test to indicate the end-point. The typical procedure was as follows: To 0.5 ml. of two per cent egg albumin there were added 0.1 ml. of buffer (pH 6.8), measured quantities of ferricyanidc, and 1.2 g. of gusnidine hydrochloride. After three minutes in a 37'C. bath the solution was cooled in ice water and then one dr0p of five per cent nitroprusside and one drop of twenty-seven per cent amonia added. The first tube in Wish no pink color dovclOped indicated the s-nount of forricyonide necessary to abolish all SH groups. Guanidinc twdrochloridc was node from gum'ddine carbonate in the manner described by Anson (19). Several samples were prepared, each time from 100 g. of gumidine carbonate. Except for the first preparation in which a metal propeller stirrer was used, all samples were prepared with glass stirrers. With the exception of the first sample these preparations of guenidine hydrochloride conformed to two criteria stated by Anson (19). l) "The color @vsn by eg albu- min end nitroprueside in guanidine fwdrochloride whfiwhmld not be increased if one drop of 0.1 N cyanide is present. 2) The cases amount of ferricyanide should be required to abolish the nitro- pruseide test of dcrmtured egg albumin in guanidine twdrochloride solution whether the ferricyanide is added before or thirty minutes after the addition of guanidine hydrochloride." Anson (10) had euggeeted that when theee criteria are not wet it in probably due to utenic inpuritiee in the guanidine ludroehioride which prouote oxidation of protein eulthydryl groups by oxygen in the air. When guanidine ludrochloride wee need ee a denaturing agent, poteeeiua ferricyanide eolution wee uaed ae a titrating agent for eulthydryl groups with nitroprueeide ae the indicator of the «n- point. Accordingly, the concentration or the potassium terricyenide had to be accurately Imam, the concentration of the etock colution being determined by a method given in Headwen and Hall (21.). For actual uee in theee titratione 0.25 ul. of 0.1.0 I! potcceiun ferri- cyanide eolution wee diluted to 50 ml. to make a 0.002 ll eolution. 3. Sodium Lauryl Sulfate. A detergent, eodiun lauryl sulfate, prepared by the Amend Drug and Chuical Cmpany, 117-119 Eaet 2A“ 8t., New York, 10, NJ. we: need u the denaturing agent in nan: experimente. This val made up at rcoa temperature ae a ten per cent stock eolution. In referring to it hereafter the more exact tern eodiun dodecyl eultate will be ueed. Anecn (19) had ueed Wm). H: which, although coneieting nainly of the on compound, eodiun dodecyl eultate, wee a airture er the 010-013 coupeunie. It nae felt that the more homogeneoue product vculd be acre eatiei’aotory than Duponol PO. 1.. Heat. In addition to the methode utilizing urea, guenidine hydrochlo- ride and eodium dodecyl eulfete ae denaturing agente which keep egg 15 albumin in eolution, Won (28) developed a heat and acid denaturing procedure (which did not coagulate egg albumin) that revealed an many an groupe ae the chemical denaturante. In thie procedure 1 all. of 1.0 l hydrochloric acid uae added to eix ll. of two per cent egg albuain which m then pun-a in a so’c. bath for an we». The mixture wae cooled in ice water, a elight exceee or 1.0 II eodiua hydroxide added, and finally diluted to one per cent concmtration of egg albumin. The eulthydryl mm were detected by adding ferri- cyanide to buffered one ml. aliquote of the diluted mixture and after five ninutee at 37°C., developing Prueeian blue in the usual manner. 16 RESULTS - PART CHE l. Denaturation.by Guanidine Hydrochloride. he determine the effect of sugars, egg albumin solution was made 2 h with respect to either glucose or fructose and guarddine hydro- chloride was used as the denaturing agent. The procedure was then the same as described previouslyw Neither of these sugars had any effect on the quantity of ferricyanide needed to abolish the sulf- hydryl groups revealed by guanidine hydrochloride. The titration was the same whether egg albumin alone or egg albumin which had been made 2 h with glucose or fructose was used. Storage of the egg albumin and sugar mixture overnight in the refrigerator did not change the titration value. Titration of an amount of cysteine hydrochloride equivalent to one ml. of one per cent egg albumin gave the same value as for egg albumin alone. Since previous workers had indicated that fructose and glucose *were the most effective sugars inhibiting the denaturation of egg albumin by heat.ths eXperiments with guanidine hydrochloride were not continued with other sugars. 2. Denaturation by Sodium Dodecyl Sulfate. The eXperiments in which sodium dodecyl sulfate was the denaturing agent used the productiOn of Prussian blue as the means of analysis. In the earliest eJcperiments a direct comparison between 1? egg albumin in the absence of sugar was made with egg alan in the presence of sugar. Il‘he total values of reagents at the time of the reaction with ferricyanids was [”0 ml. Experiments were carried out with 0.2 h, 0.5 h and 1.0 M glucose and 0.5 H and 1.0 h fructose at the time of the ferrlcyanids reaction. Fifty mg. of sodium dodccyl sulfate and 0.01 mm of ferrieyanide were added. The reaction was allowed to proceed for about one and one-half minutes before 1.0 h sulfuric acid was added. A blank containing egg albumin and sugar but no sodium dodecyl sulfate at the time of the ferricyanide reaction was used. The values obtained for egg albumin and sugar were the same as the values obtained for egg albumin alone. In every case, however, in the presence of sodium dodecyl sulfate alone only about eighty to eighty-five per cent of the sulfhydryl groups that should have been present were revealed. This amount was not increased when the time of reaction with ferricyanide was increased to five minutes. Although these results are low according to Anson's standards, Mirslq (26) reported this quantity of sulfhydryl groups utilizing essentially the same methods used by Anson. Anson (27) in consenting on this report said that he has ”Found an occasional ample of recrystallised egg albumin which had a low SH content.“ The fifty ng. of sodium dodecyl sulfate, which was used in the eXperimente above, was not only enough to denature egg albumin but also to keep it in solution when acid ferric sulfate was added at the end to develop Prussian blue. The question arose as to whether 18 smaller anmnts of sodium dodccyl sulfate, which would still canpletely denature egg albumin alone, might permit the protective effect of sugar to be exhibited. Mex-intents to determine the least amt of sodium dodecyl sulfate which would completely denature egg albumin showed that twelve mg. was the smallest quantity that could be used in a total volume of 1.7 ml. at the time of the ferricyanide reaction. Additional detergent was added after the reaction had been stopped with acid to keep the egg albmrin in solution. Ten minutes reaction time with 0.025 m}! of ferricyanide was allowed. The concen- tration of sugar at the time of the ferricyanide reaction with SH groups was approximately 0.6 PI. Neither glucose nor fructose affected the amount of denatured egg albumin. It became apparent in these experiments that the sugars as well as egg albumin were reacting with ferricyxmide. Some experiments were then carried out in which tubes containing 1.0 ml. of ferro- cyanide (the equivalent of l ml. of l per cent egg albumin) and sugar could be compared with tubes containing egg albmxin and sugar. Ten mirmtes reaction with 0.025 nah of ferricymide in a total volume of 5.031. and a concentration of glucose and fructose of l K was allowed. No protective effect was dcnonstrsted whether the mint of sodium dodecyl sulfate present was 50 mg. or 12 mg. m. reaction of ferricyanide in alkaline medium with reducing sugars is the basis for their quantitative measurmt. This reaction apparently still proceeds to a very snail extent at pH 6.8. In an l9 effort to reduce this reaction without affecting the reaction between ferrlcysnide end sulfhydryl groups, a buffer pH 6.3 and only 0.002 m. of ferricyanide were used in a total volume of 5.0 ml. A reaction time of two mimtes proved to be sufficient for measurement of all SH groups in egg albumin. Experiments were then carried out in which tubes containing all reagents except egg albumin but with sugar could be used as blanks for tubes containing egg albumin with mgar. Blanks containing all reagents with egg albumin and sugar but no detergent until a ferricysnide reaction had been stopped were also used. The sugar concentration at the time of the ferricyanide reaction was 1 )4. Again, neither glucose nor fructose nor sucrose, had any effect in protecting egg albumin against denaturation by sodium dodecyl sulfate as assured by the amount of sulfhvdryl groups revealed. The concentration of egg albumin in the experiments imnediately above was only 0.2 per cent at the time of reaction with ferricysnide. Experiments were next carried out in which the concentration of egg elbwnin was 1.0 per cent, sugar was 1 K with 10 mg. of sodium dodecyl sulfate end 0.002 at! of ferricyanide in a total volume of 1.0 ml. A reaction time of five minutes was used. Practically no reaction between ferricyanide and reducing sugar occurred under these circum- stances. Emerimsnts with glucose, galactoee, and sucrose showed that none of these sugars in a concentration of l )1 inhibited the denatu- ration of egg elbtmin. 20 3. fluctuation by fleet nnd Aeid. The fir-t acid-heat deneturction experimente were cnrried out in en open teet tube with occuionel ehnking. The full amount of cult- hydryl group. wee not detectable, however, end it we found thet ell the ulthydryl grcupe could be detected regularly it the deneturetion wen curried out in e Thunberg tube in which the etncephere wee repleced with nitrogen. This procedure we ccrried out with egg elhmin node 1 u with glucoec. thie cede the commtrction of mu about 0.86 N ct the time of denaturcticn. Under thece cirmnmntcnceo only ebout enemy-nine per cent of the culfhydryl groupe were revealed. When the procedure wce carried out with e 1.0 )4 glucose concentration ct the tine of denaturcticn, sixty-nine per cent of the eultrvdryl group: were revealed. In order to be cure thct thie reduction in the amount of eulrm'dryl groupe was not due to oxidation tnking plcce during the dcmturing procedure one :1. nemplee of acid- heet denatured e33 albumin were ellowed to renct with terricycnide in the preeence of 0.5 ll. of ten per cent eodiun dodecyl eulhte. the mat of culfhydryl we. then the once u for one 1.1. of one per cent egg albumin denatured on); with ecdiun dodecyl eultete. Th1. lcditied acid-heat dcncturcticn wee carried out in the preeence of l M galectoee, l n fructooe, end 1 x eucrcee, the concen- trctione preoent when the aid-heat dcncturction woe ecccmpliched. The everege mount of eulnzydryl groups mulled in the preeence of eech of theee more ie no follow" 1 ll nlectoce 70 per cent, l M fructose '72. per cent, 1 M sucrose 72 per cent. 21 17133 US$10}! -I-"AI?.T ONE DISCUJSIOfl‘o PART OfiE In all merimenta rcrricyanide reacted with sulfhydryl groups to form ferrocyanide. In some experiments the ferricyanide ion reacted in a small degree with the reducing sugar present forming more terrocyanide. than the amount of sulflwdryl groups was deter- mined by develcping Prussian blue from ferrccyanide, the undesired action between ferricyanide ion and sugar could be compensated for by blanks containing sugar but no egg albumin. then the sulfhydryl groups were determined by using terricyanide as a titrating agent with the end-point being indicated by a negative nitroprusside test, av reaction between terricyanide ion and sugar occurring at the same time could not be measured. Thus, it was possible in experiments with guanidine hydrochloride as denaturing agent that the sugars could have prevented some denaturaticn but it was undetected because some ferricyanide reacted with sugar in a side reaction. It seems highly unlikely, however, that the reaction of ferricyanide ion with sugar would be exactly equivalent to the number of sulfhydryl groups protected by the sugar. This is indicated particularly when it is noted that fructose reacts more readily with terricyanide ion than glucose but prevents about the same ammt of denaturaticn. Yet in the presence of either sugar, titration tor sulmydryl groups in egg albumin was the same as for egg albumin alone. It was concluded, 23 therefore, that sugars did not prevent egg alum-sin dcnaturation brwg‘at about by guanidine hydrochloride. Exhaustive uperinentation failed to downstate any protective effect by glucose, fructose, galactose, or sucrose against denatu- raticn brought about by sodium dodecyl sulfate even when the mount of denaturing agent was at a minim and the concentration of sugar was as high as l M. Advantage was taken of this fact in the aperi- nents with heat denaturation in the presence of sugar to show that the decrease in sulfm'dryl groups measured was caused by inhibition of denaturaticn and not by air ouddaticn of these reactive groups. Thus aliquots of every egg albumin solution denatured by heat in the presence of sugar were subjected to further denaturation by sodium dodocyl sulfate. If the full number of sulfhydryl mups then could be detected in these aliquots it was a clear indication that no reactive groups had been lost through air oxidation in the denatur- ation procedure and subsequent handling. Therefore, an diminution of eulfliydryl groups detected in heat denatured egg albumin was the result of the protective effect of the sugar present at the time of denaturation, not the result of air oxidation of sulftwdryl groups during the denaturation procedure. In the first heat denaturation experiments the procedure described by Anson (28) was carried out in an open test tube with occasional shaking during the heating period. Subsequent measurement of the eulfhydryl groups in a one ml. aliquot of the solution never 21. revealed the full emu-rt of sulftwdryl groups. This was unchanged by further denaturatim by sodium dodecyl sulfate prior to the addition of ferricyenide, yet denaturation by sodium dodecyl sulfate of the starting egg albumin solution revealed the proper amount of eulflwdryl groups. It was concluded that some of the sulfhydryl groups were being destroyed by air oxidation during the heat denatu. ration process. This was indicated experimentany when the heat denaturation was performed in a Thunberg tube after the atmsphere had been replaced with oxygen-free nitrogen. The full number of mlflwdryl groups was detected regularly when this technique was used. All subsequent heat denaturation experiments with sugars present were perfomed in Thunberg tubes following the latter technique. Hardt (5) showed that the length of time (. few minutes to 96 hours) that sugar was in contact with egg clbmin had no influence on the count of protection against heat denaturation. This effect was indicated in a negative way when sodium dodecyl sulfate was used as denaturant. No protection was shown whether sugar was present for a few minutes or twenty-four hours before egg dhmin was denatu- m. then egg albtsnin was denatured by heat and acid at 50’s. the effect was very evident. No difference was detected whether the egg albumin was in contact with sugar for some time before acid was added or whether the sugar and acid were edited together and thus added at the same time that the heating was begun. Thus, the protective action is shown to take place almost instantaneously even in a very 25 acid solution (pH 1.0 or less, approx. 0.11. N m1). Although one of the principal criteria of denaturation traditionally has been precipitation at the isoelectric point, the processes (denaturation and precipitation) are separable. In the work of Duddles (3), which depended on coagulation of egg albumin at the isoelectric point (p8 l..8) as a quantitative msure of denatu- ration, it was a question whether sugars prevented heat denaturation or prevented the precipitation of otherwise denatured egg albumin. Since egg albumin heated in the presence of sugar did not precipitate when the sugar m dialyzed out of the solution, the evidence indicated that sugars did prevent denaturation. Hardt (h) utilised another criterion of denaturation, the appearance of sulfhydryl groups, to indicate even more definitely that sugars prevent heat denaturation both at pH [”8 and pH 8.6. The appearance of sulfhydryl groups is one of the earliest detectable changes in the denaturation of egg albumin and therefore is a more unequivocal indication of denaturation than is coagulation. The methods for measuring sulfhydryl groups utilised by hardt indicated only half the sulmydryl groups that noon (9) later showed to be present. Utilizing a method developed by Anson which reveals all the sulfhydryl groups in egg albumin, the present work has demonstrated that in the presence of l H concen- tration of several sugars at pH 1.0 only about seventy per cent of the available sulfhvdryl groups are revealed. Thus, the present work and previous work in this laboratory have shown that the 26 protective action of sugars against heat densturation of egg albmnin is demonstrable from pf! 1.0 to pH 8.6 at temperatures for any specific pH which are sufficient to completely denature the protein in the absence of sugar. That there is an association between sugars and egg album-in which stabilizes the native molecule toward heat seems likely. Such an association has not been demonstrated directly, however, and if there is one it is sufficiently weak so that it cannot be demonstra- ted in the presence of twelve mg. of sodium dodecyl sulfate, the minimum amount necessary to bring about complete denaturation of egg albumin. Neither can an association between sugar and egg albumin be demonstrated in the presence of six M guanidine tmlrochloride. It is not surprising, perhaps, that sugars are not effective inhibitors of denaturation by guanidine hydrochloride when it is noted that fatty acid anions (particularly C7 and C3 chains) which were indicated by Boyer, Ballou, Luck and coworkers to have consider- able stabilizing action in low concentration against heat denature- tion of serum albumin (13) were completely ineffective in the presence of 6 h guanidine hydrochloride (11.). Further, Boyer, gt 5;. (13) found that sugars and alcohols were among compounds which had only slight stabilising preperties for scum albumin against heat. On the other hand, preliminary experiments showed that although egg albumin was protected in some degree by caprylate in low concentrations other proteins were rendered more heat labile in the presence of caprylate. The work of luck gt g. (13, 15, 16) may partially explain why sugars are ineffective against the denaturing action of sodium dodecyl sulfate. Their uperiments showed that at very low concen- trations the effectiveness of fatty acid anions as stabilizers of serum albumin increased as the chain length increased up to C12. They were able to show that this effectiveness was paralled by an increased affinity of the longer chain fatty acid anion for scrim albumin, and at higher concentrations, where the maximum stabili- sation is produced by c7 and CB acids, the protective effect of sodium dodecyl sulfate has been supplanted by its denaturing action and ability to keep proteins in solution. The affinity of long chain fatty acid anions for proteins appears to be due to both the negative charge and the non-polar chain and is so great that it cannot be replaced in any noticeable way by the relatively weak non-polar affinity of the sugars. EXPBP EDITH. - PART TWO 28 EXPEPJI’INI‘AL - PART 'I'a'O Since the present investigation and previous work have indicated that the inhibition to denaturation by sugars occurred over a wide pH range and required only a short period of eXposure to the specific sugar, it seemed possible that there was a definite combination between egg albumin and the sugar. It was thought that such a definite combination might have electrOphoretis properties different from those of native egg albumin. Several investigators (17, 18) already have shown that a number of detergents show definite complex formation which can be demonstrated electrophoretically with various proteins including egg albumin. These detergents are charged molecules but the complexes have electrephoretie mobilities in between that of native egg albumin and that of pure detergent. since such was the case it seemed reasonable to suppose that a definite combination between egg albumin and sugar might result in a slower moving fraction, since mgsrs are not charged and do not migrate in an electric field. EMERIM The shaded. reagents and the egg albumin used in the eXperio nents described in this section were the same as those described in Emerinmntal-Part One. 29 SCLUTI KS FOR ELT'IEOPHORFSI Native egg albumin, mixtures of egg albumin and a sugar, and egg albumin heated either alone or with a sugar or related compound at 50°C. for various periods of time were examined electrophoretical- 11 at one or more of three different pH levels. The pH levels and buffer systems were: pH 5.1, 0.031. M acetic acid-0.1 H sodium acetate; pH 6.8, 0.025 M monosodiun phosphate-0.025 M disodiun phosphate; pH 3.0, 0.5 H glycine-0.1 H hydrochloric acid. Ionic strength was kept constant at 0.1 and all experiments were performed at a temperature of 1°C. In these experiments in which egg albumin was heated for various periods of time with a sugar, the concentration of sugar indicated is that present at the time of heating. Except for these experiments marked, no dialysis (see below), the amount of sugar present during the electrophoretic analysis was negligible. Approadnately two ml. of egg albumin mixture was usually diluted to ten ml. with buffer before dialysis was begun. The protein solution was then dialyced at room temperature against 100 ml. of buffer for one and one-half hours followed by dialysis against two fresh 100 ml. portions of buffer for three hours each. The dialysis was carried out in a cellophane bag made from one inch tubing (Visking Corp.) with either the bag or the buffer container turning slowly. Finally 200 ml. of buffer was added to the third 100 ml. portion of 30 buffer and the dialysis continued evemight in the cold 1‘00. (Br-5.0.) without stirring. This buffer was then used to fill the electrode vessels. The conductivity of both the buffer and the protein solution were measured and found to be approximately the same. Although a definite combination between egg albumin and a sugar might exist in the presence of an excess of the sugar, such a combination night decompose if the excess sugar were removed. Since sugars readily dialyse, the protein-sugar mixtures in some experi- ments analysed electrophoretically at pH 5.1 and pH 6.8 were not dialyaed. These mixtures were made with buffer of twice the usual concentration so that the final solution had an ionic strength of 0.1. 11:... experiments are indicated by the notation, no dialysis. No nitrogen analysis was made on the final protein solutions which were examined electrophoretically at pH 5.1 and pH 6.8. Since the concentration and volume of the initial egg albumin solution and the volume of the final solution used for electrOphoresis were known , the approximate protein concentration could be estimated. The protein in one milliliter aliquots of the final solution examined electrophoretically at pH 3.0 was precipitated by the use of one milliliter of twenty per cent trichloracetic acid. After thorough washing with ten per cent trichloracetic acid the precipitate was analysed for nitrogen by a semi-micro KJeldahl method (22) and from these results, the protein concentration was calculated. Since the buffer at pH 3.0 contained a relatively large amount of glycine, it 31 was necessaiy to separate the protein from the buffer before mining a nitrogen magpie to determine protein comentrzition. The iiolisch test using tit-{moi (29) was made on several solutions as a qualitative test for the presence of reducing sugar. arm sum-1.16 b.. o ,I .‘.4 - Q» The electrophoresis apparatus used in those exmriments was an analytical moving-boundary instmment, Model 33, made by the Perkin- Elmer Corporation. This instrument contained the usual three section electrophoresis cell with 2 x 15 x 50 m. chatmcls which requirw about two milliliters of protein solution. The schliercn seaming system of Longsworth (30) was used to record the moving boundaries. Both electrode vessels were cpen to the atmosphere. A concise description of the general experimental procedure is axon by Alberty (31). The apparatus was new when the experiments at pH 5.1 and pH 6.8 were made and a conductance cell capable of being used with the small quantities of protein needed for the clectmphoresis cell was not yet wailable. Since the conductame of these solutions was not measured neither the electric field strength nor the mobility of the boundaries could be calculated for echriments nude at pH 5.1 or pi! 6.8. RESULTS - PART THO 32 RESULTS - PART TKO E133 TROPII’YH’EI Both Riddles (3) and Hardt (1.) have shown that sugars protect egg albumin against heat denaturation at pH 5.3, the isoelectric point of egg albumin. Therefore the first attempt to demonstrate a definite combination between egg albumin and sugars by electro- phoretic analysis was carried out at a pH near the isoelectric point, that is, pH 5.1. Figure I shows comparable patterns of egg albumin alone, a sixturo of egg albumin and 0.1 M glucose and 0.1 H glucose alone. The concentration of sugar was that present during electro- phoresis. Although the patterns show the usual small inhanogenity of crystalline egg albumin, the only new peak is one caused by the diffusion of glucose at the initial boundary. The patterns give no indication of a combination between egg albumin and glucose which has a different olectrophorotic mobility from egg albumin alone. ELEQXROPHMV Q Lt. 2g élg It is well known that two protein components which are electri- cally inseparable at one pH can often be separated at another pH. The next attempt to demonstrate a definite combination between egg albumin and sugars by electrophoretic analysis was carried out at Figure 1. Electrophoretic Patterns of Native Egg Albumin, Egg Albumin with Glucose and Glucose (pH 5.1; 0.1 M ecctate buffer; (/2 I 0.1 Ascending Descending ¢————_ Native Egg nun-n. (13,000 secs. 3 csnc. ca. 1:) E Native kg Albumin in 0.1 H Glucose. No Dialysis. (15,183 secsq csnc. ca. 1.0!) L Glucose Chly, 0.1 H. No Dialysis. (15,183 secs.) 33 31» pH 6.8. Figure 2 shows comparable patterns of egg albumin alone, a mixture of egg albumin and 0.1 H glucose, and a mixture of egg albumin and 0.1 14 glucose heated at 50°C. for .1: hours. The amount of sugar given was that present during electrophoresis. Again the small inhomogenity of crystalline egg albumin at intermediate pH values and the peak caused by the diffusion of glucose at the initial boundary is shown. No new peak indicating a combination between egg albundn and glucose is visible, however. Figure 3 shows compar- rable patterns of egg albumin heated at 50.0. for six hours with 0.1 H glucose at pl! 6.8, with 2.0 H glucose at pH 3.15 and 1.0 M sucrose at pH 6.8. The snmnts of sugar indicated were present at the time of hosting, no sugar was present during electrOphoresis. Again the patterns are like native egg albumin, alone. EL! ‘0 F? a 1. Components Prodmoed by Host. Since electrOphoresis at two pH values on the alkaline side of the isoelectric point of egg albumin showed no separation of a possible sugar-protein combination the next experiments were made at pH 3.0. Figure I. shows patterns of native egg albmdn, egg albumin, pH 3.0, heated alone and with 2.0 H glucose for twenty IIimtes at 50°C. Figure 5 shows patterns of egg albumin, pH 3.0, hosted with 2.0 M glucose for one and one-half hour, three, and six 35 Figure 2. Electrophoretic Patterns of Native Egg Alb'unin, Egg Albumin with Glucose, lgg Albumin Heated with Glucose. (pH 6 .8; 0.025 n paupmto buffer; V2 s 0.1) Ascending Dose-ding L. A native Egg Alb-is. (7030 secs.3 cenc. ca. 1.01) M M Native Egg Albumin in 0.1 M)G1ucose. No Dialysis. (6300 secs.; cone. ca. 1.0! .M M Egg Albumin Heated at 50 C. for 6 Hrs. in 0.1 M Glucose. No Dialysis. (5300 secs.; cone. ca. 1.0%) 36 Figure 3. Electrophoretic Patterns of Egg Albumin Hosted at 50°C. for 513: Hours in the Presence of Glucose and Sucrose at Different pH Values. (pH 6.8; 0.025 phosphate buffer; '72 8 0.1) Ascending Descending "F Albunin Heated at pH 6.8 in 0.1 H Glasses. (000 secs.; oeee. ca. 1.0%) ”f i Alia-in Heated at pH 3.15 in 2.0 M Glucose. ( sees.) conc. ca. 1.0%) ”f MM 308?.“ at #1 6.8 in 1.0 I an"... ( secs.; one. less than 1.0!) 37 hours at 50'C. Figure 6 shows patterns of egg albumin, pH 3.0, heated with 2.0 h glucose for twenty-four hours at 50°C. and the supernatant and precipitate of such a mixture after adjustment to pH 5.8. These patterns indicated that heating egg albumin, pH 3.0, at 50°C., whether alone or with 2.0 H glucose, resulted in two components. The presence of glucose, however, altered the quantity of the two components produced. Thus egg albumin heated alone for twenty minutes resulted in a high fast moving peak and a low slow moving peak. Egg albumin heated with 2.0 H glucose for twenty minutes resulted in a high slow'noving peak. As the period of heating was increased the height of the fast moving peak increased until at twentybfour hours the patterns were much like those of egg albumin heated alone for twenty minutes. Although the fast moving component appeared to have the same mobility as native egg albumin, separation of the components of a heated egg albumin-glucose mixture by adjustment to pH l..8 showed that the fast moving component is the precipitable component and, therefore, is denatured egg albumin. The slower component remained in solution at pH i.8 and presumably is native egg albumin. Table I gives the nobility of the components shown in Figures A, 5 and 6. The protein solutions which yielded the electrophoretio patterns given in Figures A, 5 and 6 were tested for the presence of sugaery the qualitative Holisch test (29). The results for native egg albumin and egg albumin heated with 2.0 H glucose for one and one-half 38 ”are A. llaetropherotie Patterns of Native Egg Albulis, lgg nam- heated at 50‘s. 1.:- Twenty rune... “he ad with Glucose. (p! 3.0, 0.5 w glycine-0.1 w ml buffer, I]: - 0.1) Ase-ding Descending lativofiilln-in. (m ssos.; 6.31 volts/cm; conc. 0.fi1) Egg Albumin Heated at pH 3.0. (9000 socs.; 6.30 volts/cm; 1.09%) 4r- fi Egg Albumin Hosted at pH 3.0 in 2.0 M Glucose. (9000 secs.; 6.27 volts/cm; cone. 0.76%) Figure 5. Electrophsrotic Patterns of Egg Albunin hosted at 50.0. for Various Tinss in 2.0 H Glucose. (pH 3.0; 0.5 u glycine—0.1 w an buffer; 72 - 0.1) Ascending Descending. A f Ninety Hinuto Heating at pH 3.0. (9000 secs.) 6.30 volts/cm; cone. 0.795) ‘___.__. Three Hour Heating at pH 3.0. (9“!) secs.; 6.27 volts/cm; csnc. 0.76%) Six Hour Heating at pH 3.0. (9000 secs.; 6.1.2 volts/cm; conc. 0.97%) LO Figure 6. Eloctrophoretic Patterns of Egg Albumin Heated For 'hventy-fcur Hours at 50°C., pH 3.0, in 2.0 M Glucose, and of the Supernatant and Precipitate Resulting Upon Adjustment to pH l..8. (pH 3.0; 0.5 x glycine-0.1 M 331 buffer; r/2 :- 0.1) Ascending Descending ‘___..___._. 'hvsnty—fsur Hour Heatin at pH 3.0. (9(XJO socs.; 6.29 voltsficmq cone. 0.76%) st- hponatant at pH l..8. (“1!) secs.; 6.29 volts/cm; conc. 0.32%) g ‘ Precipitate at pH l..8. (9000 socs.; 6.29 volts/cm; conc. 1.03%) new I no: mosm'rr 0? ms commune Sham m Houses 1., 5 AND 6. material a Treatment nobility (cm2 uvolt‘losoc.'1o10"5) Ascending Descending Slow Fast slow Fast Native Egg Albumin +6.30 e5 .83 ”03M 20 Min. +6s00 "6o38 ¢5e29 ¢5s82 2.0 H Glucose . Heated 20 Fin. 6.12 6.1.7 5.73 2.0 K Glucose Heated w Min. 5o73 6sl7 5o38 5.87 2.0 h Glucose Heated 3 mr. 5s50 6o12 3s95 sou 2.0 H Glucose Hosted 6 hours 5.73 6.38 545 6019 2.0 h Glucose Heated 21. Hours 5.65 6.1.0 5.39 6.18 Supernatant 5.69 5.1.8 hour and twentybfour hours were negative. Egg albumin hosted with 2.0 M glucose for twenty minutes, three hours, and six hours gave a trace test. The last buffer solutions against which these protein solutions had been finally dialyzed all gave negative tests. These results indicated that a protein-glucose combination stable to dialysis was not formed. 2. Separation and Study'of Components Produced by Rent. To indicate more definitely whether the two components found when egg albumin was heated alone were the same as the two components found when egg albumin was heated with 2.0 H glucose, the electro- phoretic runs shown in Figures 7-10 were node. The mobility of the components found in these runs is given in Table 11. Figure 7 shows patterns of native egg albumin and egg albumin, pH 3.0, heated at 50°C. for twenty'minutes. Figure 8 shows patterns of the precipitate and supernatant resulting when the heated egg albumin was adjusted to pH h.8. These patterns together with the mobilities given in Thbls II show that when egg albumin, pH 3.0, is heated at 50°C. the resulting fast component is precipitated at pH h.8 and therefore, is denatured egg albumin, while the slow component is soluble at pH 5.8 and presumably is native egg albumin. This is true in spite of the fist that the precipitable fast component has the same mobility as the initial native egg albumin while the soluble slow component moves more slowly than the starting material. Figure 9 shows patterns of egg albumin, pH 3.0, heated with 2.0 H glucose at 50°C. for six hours L3 Figure 7. Kloctrophoretic Patterns of Native Egg Albumin, and Egg Albumin Heated for 20 Minutes at 50°C., pH 3.0. (pH 3.0; 0.5 n glycine-0.1 :1 m1 buffer; :72 - 0.1) Ascending Descending .5 A Native Egg Albumin Without Heat. (9000 socs.; 6.1.0 volts/cm; conc. 0.93%) L-A Egg Albunin After Heating. (9000 socs.; 6.35 volts/cm; conc. 1.27%) Figure 8. Electrophoretic Patterns of the Precipitate and of the Supernatant After Adjustment to pH 1..8 of Egg Albumin, Heated for 20 Minutes at 50°C., pH 3.0. (pH 3. 0; 0.5 M glycine-0. 1 )4 m1 buffer; Ascending 72 a 0.1) Descending L H: Precipitate at 1..8. (9000 socs.; 6.31. volts/cm; cone. 0. 61.%) JL .1. supernatant at pH 1.. 8. (9000 secs.) 6.39 volts/cm; conc. 1.07%) h 1.5 Figure 9. mectrophoretic Patterns of Egg Albumin Heated For Six Hours at 50°C., pH 3.0, in 2.0 M Glucose, and of the Supernatant and Precipitate Resulting Upon Adjustment to pH 1..8. (pH 3.0; 0.5 M glycine-0.1 )4 m1 buffer; 72 = 0.1) Asc ending Descending 1... as, Egg Albumin and Glucose. (9000 secs.; 6.22 volts/cm; cone. ca. 0.6%) 1... as Supernatant at pH 1..8. (90(1) socs.; 6.1.0 volts/cm; conc. 0.66%) Not Recorded Precipitate at pH 1..8. (9000 secs.; 6.1.0 volts/cm; conc. 0.83%) [.6 Figure 10. Electrophoretic Patterns of Egg Albumin Heated For Six Hours at 50°C., pH 3.0, in 2.0 M Glucose, tad of the Supernatant cad Precipitate Resulting Upon Adjustment to pH h.8. (pH 3.0; 0.5 M glycine-0.1 M an buffer; 7/2 II 0.1) Ascending Descending L m. Egg Albumin and Glucose. (90(1) secs.; 6.33 volts/cm; conc. 0.92%) ‘_____,- Precipdtste st pH h.8. (m .“.e‘ 6.% VOlta/cme‘ cones 0e36z) g A Shpematant at pi m8. (90(1) secs.; 6.38 volts/cm; conc. 0.81%) 1.? T534213 II THE MOBILITY OF THE. CCéiPxfiéJqTS Slick?! IN FIGL—‘REZS 7-10. mam-m a. Treatment mummy (cm? wolt‘loseo 31.195) Ascending Descending Slow Fast Slaw Past Native 2‘35 15.1me +6.12 +5.75 Heated 20 l‘in. +5 .65 6.16 «16.02 5.57 Precipitate 5.59 6.06 6.93 5.1.9 mpermtmt 5.69 5.05 5.93 fiégtgd??g;s 6.11. 6.68 5.66 6.32 mpematzmt 6.02 5.60 Precipitate 6.1.0 fifié‘fifig 5.1.5 6.06 tun 5.62 Mimi-bate 5 059 6 033 5 .0. 5 072 mpematant 5.70 A.80 L8 and of the supernatant and precipitate resulting when this mixture was adjusted to pH he. Again the fast component is precipitated at the isoelectric point of egg albumin while the slow component remains in solution. Because the mobilities of the compensate shown in Figure 9 are different from those shown in Figures 7 and 8, a second electrophoretic pattern of egg albumin, pH 3.0, heated with 2.0 )1 glucose at 50‘s. for .1: hours 1. given in Figure 10. m. mums» of the «exponents shown in Figure 10 are approximately the same as the mobilities of the components shown in Figures 7 and 8. Although the mobilities from one electrophoretie run to another are not constant, the results given in Tables I and 11 show, in general, that the presence of glucose does not change the nobility of the two components resulting when egg albumin is heated. 3. Effect of M algars, Two Polyols and Glycine on the M Cuponents. Figure 11 shows patterns of egg albumin alone, pH 3.0, heated at 50 C. for six hours and for twentybtour hours. Figure 12 shows patterns of egg albmnin, pH 3.0, heated idth 2.0 M glucose at 50'C. for six hours and for twenty-four hours. Figure 13 shows patterns of egg albumin, pH 3.0, heated with 2.0 H namitol at 50°C. for six hours and for twentyh-four hairs. These results indicate that glucose and the somewhat related polyol, mannitol, have about the same pro- tective effect for egg elbxmin against heat denaturation. Figure 11. shows patterns of egg albumin, pH 3.0, heated with 2.0 H. fructose 1.9 at 50°C. for six hours and for twenty-four hours. These results show that fructose does not protect egg albumin against heat donaturation as well as glucose or mamdtol. The patterns after heat for six hours with fructose are about the same as after heat for twenty-four hours with glucose. Figure 15 shows patterns of egg albumin, pH 3.0, heated with 2.0 H glycine at 50°C. for six hours and for tmtybfmr hours. These patterns show only one principal component and, since it is a slow moving component, this is taken as evidence that glycine completely protects egg albumin against denaturation under the circumstances of these experiments. Figure 16 shows patterns of egg albumin, pH 3.0, heated with 2.0 1: glycerol at 50°C. for .1: hours and twenty-four hours. The patterns are almost identical to those shown in Figure 15 thus indicating that glycerol offers practically no protection against heat denaturation as carried mt here. The mobility of the components shown in Figures 11-16 are given in Table III. Figure 11. Electrophoretic Patterns of Egg Albumin Heated Alone for 6 and 2h Hours, at 50°C., pH 3.0. (pH 3.0; 0.5 M glycine-0.1 M an buffer; r/2 - 0.1) Ascending Descending W 4— -J_\ T—' Six Hour Heating. (9000 secs.; 6.h3 volts/cm.; conc. 1.32%) if h u-J), f Twentybfour Hour Heating. (9000 secs.; 6.Ll volts/cm.; conc. 1.21%) 51 Figure 12. Electrophoretic Patterns of Egg Albumin Heated For 311 and hunty-rour Hours at 5o'c., pH 3.0, in the Presence of 2.0 H Glucose. (pH 3.0; 0.5 H glycine-0.1 M an buffer; r/2 s 0.1) Ascending Descending ‘—.__._._.._— Six Hour Heating. (9000 secs.; 6.35 volts/cm.; conc. 0.86%) Twenty-four Hour Heating. (9000 secs.; 6.38 volts cm.; conc. 0.88%) 52 Figure 13. Electrophoretic Patterns of Egg Albumin Heated For Six and THentyhfour Hours at 50°C., pH 3.0, in the Presence of 2.0 H Hannitol. (pH 3.0; 0.5 H glycine-0.1 M HCl buffer; '72 = 0.1) Ascending . Descending Six Hour Heating. (9000 secs.; 6.38 volts/cm.; conc. 0.92%) ¢——._______i-o____4 Twenty-four Hour Heatin . (9000 secs.; 6.h0 voltsficm.; conc. 1.02%) 53 Figure 1h. Electrophoretic Patterns of Egg Albumin Hosted For Six and Twenty-four Hours at 50.0., pH 3.0, in the Presence of 2.0 M Fructose. (pH 3.0; 0.5 M glycine-0.1 M an buffer; ’72 - 0.1) Ascending Descending .__._- Six Heur Heating. (9000 secs.3 6.1.1 volts/cm; conc. 0.92%) ‘: ; Twenty-four Hour Heatin . (9000 secs.; 6.35 voltsficmq conc. ca. 1.0%) 5h Figure 15. Electrophoretic Patterns of Egg Albumin Heated For Six and Mnty—four Hours at 50'C., pH 3.0, in the Presence of 2.0 M Glycine. (pH 3.0; 0.5 H glycine-0.1 M ml buffer; 72 = 0.1) Ascending Descending Six Hour Heating. (9000 secs.; 6.38 volts/cm; cone. 1.07%) Twenty-four Hour Heatin . (9000 secs.; 6.38 volts7cm.; conc. 1.17%) 55 Figure 16. Electrepheretic Patterns of Egg Albumin Heated For Six and Netty-fear Hsure at 50.0., pl 3.0, in the Presence of 2.0 II Glycerol. (pH 3.0; 0.5 n glycine-0.1 s an buffer; 172 a 0.1) AsceMisg Descending h. -J\ Six Hour Heating. (9000 secs.; 6.1.1 volts/cm; cone. 1.20%) 1 A f H a... -J\ T Twenty-four Hour Heatin . (9000 secs.; 6.31. voltsficmq conc. l.21.%) 56 TABLE 111 m hosILI'rr 0? “mm consumers Show IN FIGURES 11-16. Material a 'h‘eatment Mobility (omzovolt'losec.'1010'51 Ascending Descending Slow Fast Slos' Fast Egg Albania Alone “Cam 6 MP. §5e7o 46e56 §5e27 +6.01. Egg Albumin Alone Heated 21. Hours 2.0 H Glucose Heated 6 Hours 2.0 H Glucose Heated 21. Hours 2.0 H Hannitol Heated 6 Hmrs Heated 21. Hours 2.0 H Metose Heated 6 Hours 2.0 H anteee Heated 21. Hours 2.0 H Glycine Heated 6 Hours 2.0 H mains Heated 21. Hours 2.0 H MON}. Host“! 6 Hours 2.0 H Glycerol Heated 21. Hours 5.81 6.15 6.00 1. .70 5 .92 5-55 6 .76 6.30 6.81 6 .30 7.01. 6.88 6.1.1 6.93 5.29 5.1.8 5-61. 5.56 5.61. 6-58 5 .31 5.11 5.35 6.07 6 .27 6.1.1. 5-92 6.66 6.1.7 5 .98 6.1.0 DISC US$103 - PART T130 57 DISCUSSION - PART TWO In all the runs made at pH 5.1 and pH 6.8 there was a small side boundary which was first pointed out by Icngeworth (30) as occurring in crystalline egg albumin preparations at intermediate pH values. Iongsworth, g g. (32) have shown that although there appeared to be some variability in the relative amount of the lesser component from preparation to preparation, it was found in all samples of egg albumin mined regardless of source or node of preparation. Perlman (33) has indicated that the phosphorus content of the two components is different and that the mobility differmce corresponds with this difference in phosphorus content. No other differences in properties of the two compments were found. Normally non-emailibration of the protein with the buffer by dialysis results in unstable boundaries or boundaries which do not correspond to protein components. (he new boundary was thus famed at the initial boundary site by diffusion of sugar. This has readily indicated expeMentally and such boundaries were thereafter imored whenever undialysed or incompletely dialysed couples were used. No other changes were detected. Because electrophoresis of a mixture of egg albxmin and glucose did not reveal any new protein componmt either at pH 5.1 or pH 6.8, heat was applied to some mixtures with the hope of increasing the 58 amount of the presumed egg mun-sugar component. Since the protective effect of sugars had been demonstrated only against heat denaturation there was no ezperimental indication that an egg albumin- sugar combination existed except at higher temperatures. The use of heat did not alter the pattern and the conditions under which heat was applied were so mild that no or very little denaturation may have taken place. (he of the runs was made with an egg albuadn-glucose mixture heated at pH 3.0 and 50°C. for six hours. These conditions later were shown to cause denaturation detectable with electrophoresis at pH 3.0, but electrophoresis at pH 6.8 revealed no protein-sugar component or amvthing indicative of partial denaturation of the egg albumin. This result follows the experience of several other investigators (30, 32, 3h, 35) who have shown the mobility of denatured egg albumin differs only slightly frat: that of native egg albmin. Electrophoresis at pH 3.0 revealed only one component in native egg albumin following the experience of other investigators (32). After egg albumin had been heated at 50°C. and pH 3.0 two components were visible and the experimental results (Figures 7 and 8, Table 11) show that the faster component is denatured egg albmnin and the slower component is presumably native egg albumin, although its mobility is slower than that of the original native egg albumin. The presence of sugars obviously changes the quantity of these two components resulting from a givm amount of heat, but does not alter 59 their mobility. The experiments pictured in {Figures 9 and 10 show that the faster emponent produced in the presence of sugars again is precipitated at the isoelectric point of egg albumin whereas the slower comenent remains in solution. Electrophoresis has thus become another news of indicating that wears do protect egg, albmin against heat denaturation for a time, although evmtuelly the egg albumin is denatured. Hardt (36) previously had shown that sugars inhibit or prevent the formation of the "c" cosmonaut which appears after heating bovine serum or plasma. 'me nobility of the two components is not as wsiforn as one would like to be assured that they are the same cwpononts from one run to another. Part of this non-unifomity m be due to unknown experimental errors. Another explanation is indicated by McPherson and Heidelberg" (37) who enswntered extreme difficulty in obtain- ing successive preparations of acid-denatured egg albumin with reproducible properties. Ecperisental substantiation of this expla- mtion for the variation emeuntered is indicated when it is noted that the abilities of the conponmts shows: in Figure 9 (see Table II) which came from one preparation are relatively unifors. This is tree also of another siailar preparation shown in Figure 10 (see table II). The abilities of the eupenents of the two preparatim are quite different although each was supposedly prepared in the ease sanner. Protein denaturatica in general is dependent in some degree upon hydrogen ion concentration. Steinbsrdt (38) first explained the influence of pH on the rate of protein denaturetion as being the result of the effect a the ionisation of critical groups. back, 33 g. (16) have indicated that the protective effect of fatty acid anions for some albumin against heat is due, at least in part, to an affinity for the charged groups of the albumin, a condition detected electrophoreticelly. It was shown also that the non-polar portion of the molecule use important in the stabilising action. Therefore it seemed probable that the stabilisation was the result of the attrac- tion of different groups of the albumin molecule for both the polar and non-polar portions of the fatty acid anions. Other investigators (17, 1.8) have shown a definite combination of detergents with the charged groups of protein materials. Again the non-polar portion of the molecule is important in the affinity of the compound for protein Since these materials are relatively large molecules the view often is advanced that the affinity for protein is great enough te force the protein nolecule apart at various points thus resulting in loosening of the protein structure and thereby causing denaturctim. Because the charge state of a protein has been indicated to be inportant in the denaturaticn of the material, it sewed possible that the protective effect of sugars for egg albumin against heat light be due in port to an effect on the charged group which could be demonstrated electrophoreticslly. Since sugars appear to have as effect on the nobility of egg albumin at several different p3 61 levels it met be cmcluded that the protective effect is dependent solely upon e combination with the non-polar side chains of the protein. Although such e combination has yet to be demonstrated, it some reasonable, in View of the importence of the non-polar portions of fatty ecid onions and detergents, to believe that one exists. Since only the non-polar portion of the protein apparently is involved in the protective action by sugars, their relatively low activity can be explained. Putnam (39) has suggested that the protective effect of :11ng may be due to the osmotic property of the added eohxte. However, earlier work in this laboratory (3, I.) showed substantial differences between different sugars at the same molar concentration. The present work has shown that glycerol is much lees effective than glucose ct the same molar concentration. Such evidence does not support Putnam's suggestion. Previous workers (2, 3, I.) have imiicated differencee in the protective efficiency of several sugars. The present work has danonetrated manor differences than were previously indicated, probably because of the lower pH at which the denaturation was performed. Both this and earlier work (2) indicate that glycerol is e relatively poor stabilizing agent. In fact of all the more end polyols tried in this laboratory (3, l. and the present work) glycerol has been the poorest stabilizing agent. There appears to be relatively little difference in protective action between 62 pentoees, hexoses, e natal, and a disaccharide. The apparently excellent stabilizing action of glycine probably resulted from its buffering action because in all cases, the protective compound was added to egg albumin adjusted to p!! 3.0. Sugars and polyole which have no buffering action changed the pH mly slightly. Addition of glycine probably shifted the pH upward from pH 3.0 to a pH where egg elbun‘in is stable at 50°C. C ONC LUSICXn’S 1. 2. 3. I... 5. 6. 7. 63 COfiCLUSIORS The sugars, glucose or fructose, were shown not to have any protective action against egg albumin denaturation caused by 6 H guanidine fwdrochloride. The sugars, glucose, fructose. galactose or sucrose, were chem not to have any protective action against egg albumin denatu- ration caused by s minimum amount of sodium dodecyl sulfate. The sugars, glucose, fructose, galactoee or sucrose in a concentration of 1.0 H were all shown to have s substantial protective effect against heat denaturation of egg albmzin in acid media (approximately 0.11. N hydrochloric acid). This protective effect occurs almost instantaneously. Ito combination of glucose and egg alum-in could be directly demonstrated by electrophoresis at pl! 3.0. 5.1 or 6.8. Egg albumin, pH 3.0. heated at 50°C.. either alone or with sugar, resulted in two components; one component was shown to be denatured egg albumin while the other component was shown to be native egg albumin. The stabilizing effect of glucose, fructose, manrdtol and glycine on egg albumin held at pi! 3.0 and 50"}. for six hours and treaty- four hours was demmstrated electrophoretically. No stabilizing effect by glycerol under the same circumstances could be demonstrated. LITiltATU'z-tfi CITED (1) (2) (3) (lo) (5) (6) (7) (8) (9) 6h LITHDlTUEE CITED Neuberg, C., lbdrotropieche Erecheinungen I. Nitteilung, Biochem. Ze’ 1.6., 107 (1916’s Beilinsson, A., Thermostabilieation der Fiweisslosun en nit Rohrsucker und Glycerin, Biochen. 2., a}, 399 (1929?. 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