‘ 13 i. 1 {WE MCBELITY CF ECG ALELMIJN 11V (ELL'CC E SCLL‘T’C‘N ”‘er ‘cr the Degree of M 5: =5 Rube}; 1‘ j Waited} 1m? o .J. y 3! .1" l. ' ( w..‘ v: . .u v A» ,. . .‘ .er ‘ .«1 Q. -\ i ' a. . 0 ‘ v . J ' .y ,. , I, o " l x ;( ‘d . I 0., . . A“ x? x o»; 7? ‘3 _, . 4,. THE MOBILITY OF EGG ALBUKIN IN GLUCOSE SOLUTION THESIS FOR THE DEGREE OF M. 3. ROBERT J. WESTFALL I937 5 0n... flu Auk a“, .6. . THE MOBILITY OF ECG ALBUMIN m GLUCO L1 t‘J SOLUTION A THESIS SUBEITTED TO THE FACULTY OF MICHIGAN STATE COLLEGE OF AGRICULTURE AND APPLIED SCIENCE IN PARTIAL FULFILHJEE‘IT OF THE} REQUIRE-.1338 FOR THE DEGREE OF MASTER OF SCIENCE By ROBERT J. WESTFALL June, 1937 ACKNOWLEDGEEiT The writer wishes to express sincere thanks to Professor C. D. Ball for his assistance and considerateness, which have added greatly to the pleasure of doing this work. KM; meg/W COII'ZEI‘JTS Page Introduction 1. Historical A. Methods of determining mobility . . . . . . . . l B, Previous determinations of the mobility of egg albumin . . . . . o o . . . . . o . 5 C. Relation of mobility to the zeta potential. . . 6 D. Relation of the zeta potential to stability 0 . 8 B. Effect of sugars and related compounds on eeealbumin............... 9 II. Experimental A. Description of cell and calibration . . . . . . 10 B. Determination bf mobilities . . . . . . . . . . 17 C. Determination of viscosity . . . . . . . . . . 23 III. DISCUSSIOD o o o o o o o o o o o o o o o o o o o o o o o 26 IV. COHCIUSIODS and smmary . Q . o o 9 o o o o o O O O O O 34 V. Bibliography 0 o o o o o o o o o o o o o o o o o o o o o 35 INTRODUCTION The importance of surface phenomena in biological problems can be realized from the fact that all processes of living tissues are directly or indirectly related to them. The nature of a protein surface is of special interest, since proteins are believed to be present in all living cells. One important method of investigating surface constitution involves making some measurement dependent on the potential across the phase boundary —- the "electrokinetic' potential. Determination of the mobility of a particle in an electric field is one such measurement which is applicable to a solution of a protein. The nature of the surface of egg albumin in sugar solutions is of interest because in the presence of suga s, egg alhumin is not so readily coagulated by heat or by ultraviolet light, as was shown by Beilinnson (l) and Duddles (2). Surface properties are known often to ‘.J. have a large influence on stability toward coagulation; accordingly, t is hoped t'is investigation may help to explain the mechanism by which sugars stabilize the egg albumin against coagulation. HISTORICAL The literature relevant to this problem can most conveniently be reviewed in the following order: A. Methods of determining the mobility of a colloidal particle. Results reported for the mobility of egg albumin. The relation between mobility and the zeta potential. The relation between the zeta potential and the stability of sols. The effect of sugars and related compounds on properties of egg albumin. A. Methods of determining the mobility of a colloidal particle. Three general methods have been developed for determining the mobility of a particle in an electric field. These are: 1. The transport method, in which the amount of a.substance migrating across a given plane in a field of known strength is determined by some analytical procedure. 2. The moving boundary method, which depends on the main- tenance of a distinct boundary between a solution containing the particles in question and another solution, the same except for the absence of the particles, when a known field is present at the boundary. The location of the boundary may be determined visually or photographically, and the velocity of its movement thus determined in the known field. Under the proper conditions, the velocity of the boundary is the same as that of the particle. 3. The microscopic observation method, in which a particle large enough to be seen with a microscope moves in a cell with a known field strength, and its velocity is determined by the use of a scale included in the magnifying system. In all three methods, it is obvious that the particle will migrate toward the electrode with a charge of sign opposite to its own. The first two methods are equally applicable to colloidal and crystalloidal systems; the last only to colloidal ones. Since all pro» teins are recognized to be of colloidal nature, the methods will be considered only in their application to colloidal systems. The first method has received the following comment by Abramson (3) o “In principle the transport method of Hittorf (as for simple ions) is designed to give the most theoretically acceptable values of the electric mobility of the disperse phase of colloidal suspensions and solutions o..... In practice, all of the difficulties inherent in the determination of the transport number of ions are present, in addition to those imposed by the presence of a.dispersed phase.‘ As e.result of these difficulties, the method has been chiefly applied to sols of metals, but it has also been applied to soaps by McBain and Bowden (4) and by Laing (5), and to proteins by Greenberg and Schmidt (6). These workers were interested chiefly in obtaining the transport numbers of both the '0‘... positive and negative particles present. Engel and Pauli (7) have described an apparatus, of rather complicated construction, for the deter- mination of the mobility of colloidal particles by the transport method, and have applied it to several inorganic sols. The simpler type of apparatus devised by Paine (8) and by Henry and Brittain (9) for the determination of the mobility of particles in metal sols seems to be of more general applicability. Briefly, it consists of an inverted Uetube of glass, the arms of which dip into two vessels containing platinum electrodes and enough sol to fill the U-tube and part of each vessel. The U-tube is filled by suction from.a side-tube sealed to the top of the bend, and a known potential difference is applied to the electrodes for a given time. The solution is then allowed to flow back by releasing the auction at the top. The change in concentration in each vessel is then determined, and from it the mobility is calculated by the following equation, given by Abramson (3} and Paine (8)- v :: m k (I) I. I t ' in illicit - v = mobility of colloidal particles m.=: mass of colloid transferred across boundary B'z: original.mass of colloid per cubic centimeter k = specific conductivity of solution I =: current flowing t = time current flows Henry and Brittain (9) sealed two subsidiary electrodes into the U—tube near the top of the bend, and usedthe equation - u s t (II) N H voltage between the subsidiary electrodes D =1 a constant depending only on the position of the electrodes between which E is measured The moving boundary method has been more widely applied to protein sols. Svedberg and Jette (10) applied it to egg albumin, using photography of the fluorescence in ultraviolet light to determine the position of the boundary. Later, Svedberg and.Tiselius (ll) feund absorption of ultraviolet light a more reliable criterion of the presence of egg albumin and other proteins. This method has been applied to a number of proteins by Tiselius (12}. There is still some dispute as to the relations that should exist between the intermicellar liquid of the protein sol and the supernatant liquid; Henry and Brittain (9) give an extended discussion of this uncertainty. The method of microscopic observation depends for its application to proteins on the fact that most proteins may be adsorbed on the surface of particles large enough to be seen with a microscope. Mattson (13), Northrop and Kunitz (l4), and Abramson (15) have developed this method most extensively, and its application to proteins by théfworkers-has furnished much important data. The particles employed have usually been either collodion or quartz. Since, in the small cells used, electro- osmotic flow takes place as well as electrophoresis, the motion must be observed in a particular position if it is not to be complicated by the flow of liquid. The question naturally arises whether a film so adsorbed is the same as the original protein. Abramson (15) has shown with egg albumin and serum albumin that the mobility at different ccidities is the same as obtained with the moving boundary method. It has been shown, also by Abramson (15), that when a pure protein is adsorbed on the sur- face of a particle, the mobility is independent of the size and shape of the particle and is the same as far the protein particles themselves. The film.must be of only one protein and must completely cover the particles. B. Previous determinations of the mobility of egg albumin. Hardy (16) was the first to determine the mobility of egg albumin at different acidities. Using the moving boundary method, he found that particles of heat coagulated egg white migrated to the positive electrode in alkaline solutions, to the negative electrode in fairly acid solutions, and not at all at an intermediate acidity. The method of Northrop and Kunitz (14), using microscopic observation, was applied by Loeb (l?) to particles of collodion with an adsorbed film of egg albumin; the effect of varying pH and salt con- centration was determined. Abramson (5) and his co—vorkers have made a large number of determinations by this method, using various types of cells, different salts, and even other solutions than aqueous. Smith (18,19) found with this method that the mobility of albumin-coated particles depended on the concentration of protein and of the salt present, and on the nature of the salt, as well as on the pH. Increas- ing protein concentration and increasing ionic strength both lowered the pH of zero mobility, and different ions affected it in the order of the lyotropic series. Svedberg and Jette (10), Svedberg and Tiselius (11), and Tiselius (12) have obtained results for egg albumin by the moving boundary method. Since in this method the protein isysolution in its original state, not transformed by surface denaturation or otherwise, the results provide a basis for testing the validity of other methods. No results have been reported for the mobility of egg albumin by the transport method. 0. The relation of mobility to the zeta potential. Soon after it was demonstrated that colloidal surfaces possess a charge, theoretical investigations were carried out to determine the relation between the potential of the charge and the phenomena resulting when an electric field was super-imposed on the charges. The inves- tigations of Helmholtz and subsequent workers are summarized by Abramson (5), who gives a thorough discussion of the derivation of the relations. The following equation is obtained for the mobility of a particle in an electric field - 1226;}: ; inwhich-v 4 1T n v : mobility of particle 4: 2 potential across the double layer n :: dielectric constant in the double layer X 2 field strength T) viscosity of the surrounding medium H The term.€§5 , with the subscript referring to electrOphoresis, is commonly referred to as the zeta potential. Abramson (5) points out that the equation was really derived for the case of electro-osmosis or streaming potential, and applied to electrophoresis by analogy. That the analogy is true is indicated by the work of Briggs (20) and of Abramson (21) with proteins, and of Henry (22) with waxscoated particles and surfaces. These workers found that the velocity of a liquid with respect to a stationary.surface is the same as that of'a particle, having the same surface and moving in the same liquid, when all other factors are the same. The use of the constant 4 has been questioned by Debye and Hfickel (23, 24). Later investigations by Henry (22) indicate that the value 4 is correct for insulating particles, such as exist in protein sols, when the particles are large in comparison with the thickness of the double layer; while for ions, the larger values of the constant used by Debye and Hfickel (23) are correct. The value which should be used for D is, however, less certain. By definition, it is the dielectric constant in the double layer itself. Since this is not known, the value for'the surrounding medium is used, with the assumption that conditions in the double layer do not alter it greatly, although according to Briggs (25) it is almost certain to be somewhat different. That the assumption is not unjustified is indicated by the results of Daniel (26} using solutions containing water and alcohol in various proportions. These findings indicate that the theory is correct to within ten percent if the values for D and y of the surround- ing medium be used. The validity of equation III has been questioned by McBain (27), who raises the objection that the uniform, continuous double layer postulated probably does not exist in exactly that form. However, the many relations among the electrokinetic phenomena which theory calls for and which experiment has verified, as described by Abramson (3), constitute a very strong argument for the theory. D. Relation of the zeta potential to stability. Hardy (16) was the first to postulate a dependence of col- loidal stability of the charge of the particles. Later investigations by Burton (28) and Pcwis (29) confirmed this in sons cases, and more- over showed that in these cases the sol become unstable henever the 1‘. zeta potential fell below a critical value. This is apparently con- tradicted by the knowledge that many proteins, for instance, are stable even when isoelectric, as shown by Michaelis and Rona (30) with serum albumin. It has even been found by Kruyt and van der Willigen (31) that in some cases coagulation may be accompanied by an increase of the zeta potential. This contradiction has been explained by Kruyt and his co- workers (32) as due to the hydration of the particles in the systems which are stable even when the particles are uncharged. This has appar- ently been confirmed by the work of de Jong (33), using alcohol and tannin to dehydrate the particles and thereby render them unstable. Egg albumin and other native albumins would then belong to this class, since they are readily soluble in water even when isoeleotric. Loeb (34) per- formed experiments with heatsur and surface-denatured egg albumin which show that the charge does have an important effect on their stability, since particles of collodion coated with adsorbed albumin or particles of heat-denatured protein coagulate when salts or the pH lowered the zeta potential to about twelve millivolts or less. According to the theory of Kruyt, this would show that denatured egg albumin is less hydrated than the native protein. The validity of Kruyt's theory has been questioned by Greenberg and Greenberg (35), who found that even in the case of supposedly highly hydrated sols such as casein, gelatin and agar-agar, ultrafiltration experiments showed that practically all of the water present was able to dissolve salts, urea or other crystalloids. There is no completely acceptable theory of colloidal stability, then,for systems in which the particles are stable when uncharged. E. The effect of sugars and related compounds on egg albumin. Beilinnson (l) was the first to report that the presence of sucrose and glycerol inhibited heat coagulation of egg albumin. Duddles' work (2) showed that this property extended to glucose, fructose, mannose, and mannitol, all of which protected against coagulation by heat, but in varying degrees. Against coagulation by ultraviolet light, glucose showed about the same protective power as toward heat; none of the other compounds was tried in this case. This protection has also been observed by Newton and Brown (36), who showed that sucrose and glucose prevented partially the precipitation of proteins from plant saps by freezing. Donahue (57) found that the turbidity caused by precipitation with sulfosalicylic acid was decreased by the presence of methyl ~, ethyl-, and propyl alcohols, and by glycerol and glycol; but nitrogen deter- minations on the filtrates from the suspensions showed that except in the case of glycerol, the protective effect was only apparent. A similar protective effect was observed by Teorell (38), who found that the coag- ulation by heat of serum albumin in acetate buffers was inhibited by the presence of ethyl and propyl alcohols. This did not hold true, however, with egg albumin. Experiments of a different nature indicate that glucose is either IO adsorbed on or combined with egg albumin. De Anciaes and Trincao (39) found that the reducing power of mixtures of the two was less than the sum of'their separate reducing powers, as determined by the method of Hagedorn (40). The change in reducing power was almost completely pre- vented if five percent of sodium chloride were present before addition of the sugar to the protein. This effect was confirmed by Gubarev and Moiseenko (41), who subjected glucose-albumin mixtures to ultrafiltration and found the ultrafiltrate less concentrated in glucose than the original solution prepared. The change in the concentration of glucose increased with the concentration of glucose and with the pH throughout the range 3.0-8.4. Lundén (42) reported that the sweetness of sugar solutions was decreased by the presence of egg albumin in concentrations from .0052 to 32 protein. Results that are perhaps related to these were obtained by Rafalowska, Mystkowski (43) and others. They found that arginine or tyrosine apparently combined with amylase and various other posysac» charides, especially in.the more alkaline solutions in the pH range 4 - 8. Tetrapeptides and other polypeptides containing these amino acids had the same effect, as did myosin, fibrin and other proteins which presumably contained these amino acids. The criteria of the formation of compounds were changes in optical activity and the formation of precipitates. EXPER IENTAL Preparation of egg albumin Egg albumin was prepared by the method of Keckwick and Cannon (44) using sodium sulfate to precipitate the albumin. This was chosen in preference to the betterwknown method of Sorensen, which uses ammonium 11 sulfate, because any question about the addition of ammonia nitrogen, as in the latter, would be removed. Some difficulty was experienced in following the directions referred to, with respect to dissolving the sodium sulfate and precip- itating the egg albumin. Even when the sodium sulfate was added to the protein solution in small portions, masses of crystals would settle to the bottom of the vessel, so it was necessary to stir the solution vigor- ously, but at the same time avoid beating up foam which would cause BXb cessive surface denaturation. A propeller-type stirrer worked best. It was also noticed that unless stirring was continued for at least an hour after sufficient sodium sulfate had been added, the albumin was likely to precipitate on the sides of the beaker containing the mixture as an amorphous gum, which on standing would slowly revert to the crystalline state. Six dozen eggs were sufficient to prepare 1500 cubic centimeters of a six per cent solution with a specific conductivity of lO‘br less and a pH of 4.7. Description of electrophoresis cell For determining the mobility of egg albumin in glucose it was decided that the method of Henry and Brittain (9) would be best. The presence of large amounts of sugar might interfere with the adsorption of protein necessary to the microscopic method, or in the method of Svedberg and Tiselius (ll) the sugar might absorb ultraviolet light itself and obscure the boundary. The accompanying photograph (Fig. 1) illustrates the cell, which was made from twelve millimeter Pyrex tubing. Contact between the platinum electrodes and the leads to the battery and potentiometer was made through mercury in the tubes into which the electrodes were sealed. The cell was kept filled with cleaning solution when not in us e, to insure proper drainage of the inside surface. The main electrodes, at the ends of the arms of the Untubes, were of platinum wire just long enough to go aroun the tube. A slight flange on the end of the tube kept them in place and prevented bubbles from electrolysis from getting inside the tube. The diagram in Fig. 2 shows the electrical connections used during determinations. In all essentials it is identical with tl1e circuit used by Henry and Prittain (9). The voltmeter used was a Weston pre- cision instrument with a scale len3th of fifteen centiraeters, reading up to fifteen volts. The variable resistances were the “Centralab" radio type. Cuz'rent was supplied by two radio ”B" batteries connected in series The galvanometer was of the type ordinarily used in ”student type“ poten- tiometers. Ca libretion of cell h: (3 CL In order to use equation II on page 3, the cell was calibr t with N/SO potm sium chloride. A micro-ammeter was inserted in the circuit n Fig . 2. This was also a Weston precision instrument, Ho as shown at 33 reading Up to one milli mpere on a scale oi 3hteen centimeters lon3. To [do :3 (TI) calibrate the cell, the current was set at the desired value by adjw the variable resistances Raand Ryand the corresponding voltage between the subsidiary electrodes read; this was repeated at a number of dif- ferent settings over the available range. To determine the voltage, the resistance erwas first set so as to a ford ma,imum protection to the galvanometer; this was obviousl-r obtained when the galvanometer was I q portion of the resistance. T‘e reels ta ncellz p kJ H shunted across only a an Electrophoresis Cell was thena M3 sted until there was no deflection of the alvano meter on depression of the key K,. The resistance across which the falvanometer was shunted~-or, in other words, the portion of R, which was in parallel with the galvanomete r-~ as then increas 3d, and R2. again set for no de- flection of the galvanometer. Hhen this was repeeted until the galvanom- eter we shunted around all of R, , the readin of le vol t eter was the volta3e between the subsidiary electrodes. Table I records the values obtained, which are plotted in Fig. 0. The "best straight line“ through the points was extranola ed to zero current, showing an apparent polarization of O. 2 volt at the sutsi31arv electrod1s.Henry and Brittain (9), usin: a similar procedur. with more dilute potassiun chloride obtai inei 0.1 volt. They noted that this might 1—» not be due to actual polarization, but since th s possibility involves urcerta in assumptions, it was con.idere d best in this work to treat the .'¢ r1163 aw re m D deviation as :o_arization. Cunsequently, all volta' corrected by adding 0.2 volt before us sin3 in calculations. Table I Calibration of cells Amperes " l0~a Volts between subsidiary electrodes Cell # 1 Cell # 2 0.100 0.11 0.150 0.15 0.25 0.200 0.27 0.43 0.250 0.41 0.55 0.300 0.53 0.70 0.350 0.69 0.85 0.400 0.80 1.00 0.450 0.90 1.15 0.500 1.00 1.30 0.550 1.11 1.45 0.801 1.27 1.60 0.650 1.41 1.75 0.700 1.55 1.90 0.750 1.68 $3.05 0.800 1.80 2.20 0.850 $.93 2.” 0.900 2.04 2.50 0.950 2.18 2.65 1.000 2.30 2.80 Temperature 29.4'c 30.5°C- Value of k used 0.303024 0.003064 lope of “best straight lineu +2.5O -+3.00 Value of D obtained 7.96 9.18 16 Fia- 9 v SO 1» to yo b‘s.”@ _L_ Key to symbols R. and 33- 0.5 megohm variable resistances R¢,and 34- 50,000 ohm variable resistances B,- 90 volts from radio I'B" batteries 32- 22 volts from radio “B" batteries 6 - galvanometer V - voltmeter K;- Tapping key Kg- switch E)» main electrodes 32- subsidiary electrodes AB — microammeter inserted in series for calibration r‘ .Y i 1 T Y T f ' r W’ I 7 I I 7“.- .- ~o-— I o-I~ omeéu-QI oI — I 0 ' I I - I . o . u - v - I I I -~ , .- . . - o o . . 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'I I ' I I ' ' l‘ I ‘ v I ‘ I 4—71 I, 1’ — lg - p... — -- ..— .co— - .. t—....-.—-——o - OI _- _o-I— - I - —-—- .0- .--_-———.—I——-._ I I I I o I I- I I . I I V l ' . ' . I o... I - u I ‘v | I I I . u I I . - a I . I ._. _4.-'... o ..I o- .. .. .-... .o.— c~ -—- - . 9—.-- -..— I v 0 ~ I I — I I I -I I . I. I. I . .' I 5 . . I 0 I n I — v . . I t I I o I I ' l | I . I 7 ' ' " I - t 2 >- ._—... ___.. _ ._ .-.-... ...... --I—.__..—-I._ .— ..p — — .....- .. __.-:_._... — - —. .-.-.- o.- _. ..p _- -..- ....I I ~ . - o . ~ I n I u o . . . . . . ‘ I ' 'i ' ' I ' I ° I I I I . . . I. . . I . . . . ' II . v u I I - - v 1' . - Y . _ -——-—— - o—. —o -. -.-o- -' . . - -—..o- o o 0 . ._'- o+<-' . - o-I -— -. o I o 9 —. worm-an - q I 0-1 I . ‘ II c I I ‘ I I I — I ‘ .: v I I I ' - I ¢ I 0' ' . n .u . - ~ - o - _ . l I I I I . I . I . I I I - --.———.. -——.—¢. ..————-o-- —-—-—r J— F—u—“fl—m—Lr—L—fl-fifi awn—tun?- —- u..." w—-o-—_..J I . I. a . . . .I , 'I . . . I I. I I . v. - . .1. I I. I I I - I I o . I I - I a v I l . . . I I -I . . I ' ._ ~ -- I ——I ~- - O‘--¢O - -I >- ~— I -o I— . I- o I 1 . . -l . . . | I ' I I - - I I .. ‘ . In . l I. a I 1 I - I I I . I I . I A - - - : o -m-T-‘ - I -_----—-—— - up— *‘——I. ~--—--—— quo— I I i T .- ..up—— —— - - - I - _— I . ‘ - ‘ I - ' ’ I . . . Q I .- I ' ' I n I l . I I I I ' . I I l ' I ' I I I I g I —<—qI ou-o< 0—0 1- : I - 4» - I 9 u- . - - o I I I n a - -— . I o - u :- +-‘ ‘ u q' u u v 1 n I I . I. . . . . x ' . . i . u I I o I I I. n I . I ~ I 2 i j l I ‘. 1 4 11:14 ‘1’: A ]___ .- r I ‘I T Y .— . I .‘ . I! . I .2. It. . . . . I .. I l ' I v . ' 0 ¢ ' " 0 l6 , " ’ I ' 113‘ i , . .— -— .- -o I- ....-- .. ~00- - . .- . '¢ t I I I I I I | I l . I I - ' ‘ I I ' I I l l I I I I | I I no . I I I I I I 1 . ' I I I _ -- —.—-— +.—-—— -———. .u- -O—v-‘l Y".- . -.-0-o ————— ___. -pc—u— .-—_. -—. .—.— u - —. ._ _ . -— — - o g _ - ..J . I l I 'I ' ' 7 . I I .' I I . I . . I I u I ' i ' . " I I I I I I ‘ I ' ' I . I ‘ I I 5. 1 e -.--_.i_._..,.-.-.. ...I. ..- ...., {$11193me J, . __ 1.: _ _ - -. . - . I .4 O I I 'f ' I I ‘ - I ‘ I I I I I l I I .. ....- .. .11...“ .. l . , . . p I I ' I C no; u - I I. .Q. . . I I I I I I . I l , l I r-A 2:11 to... .-1 . L .1”... . I I I. I I I I -- I I I v I I No. ZFO-F. THE H. COLE C0.. C 17 The cell constant was calculated from the equation -- DzL—Ls as given by Henry and Brittain (9), in which the symbols have the same definitions as in equations I and II, pages 3 and 4. The value taken for E was in each case the slope of the line in Fig. 3; accordingly, I became ‘3 10 . The value for k was the specific conductivity of N/SO potassium chloride at the room temperature when the cell was calibrated. Method of making determinations. In determining the mobility of egg albumin, the work of Smith (18, 19) indicates that the ionic strength must be considered. Accord- ingly, sodium acetate-acetic acid buffers were used in the same way as in their work, keeping the concentration of sodium ion constant while the pH was varied by changing the amount of acetic acid added. The solutions were prepared as follows: Assuming 100 cubic centimeters of solution was required, five cubic centimeters of M/S sodium hydroxide was pipetted into a 100 cubic centimeter graduated cysinder, and sufficient acetic acid added to give the desired pH. Either M/S or M acetic acid was used, according to convenience. Fifty cubic centimeters of 2M glucose solution was then added, or approximately that volume of water, and sufficient freshly filtered stock albumin solution to give about two per cent protein in the final solution. After dilution to 100 cubic centimeters, the solution was mixed and refiltered with as little foaming as possible, to avoid surface denaturation. Proportionate amounts were used to prepare less solution. ‘ The pH of the solution was determined with a Beckman glass electrode. The results were reproducible within a pH range of 1:0.02 ~ 0.03. ya CO A trial was made to see if the addition of glucose in the concentration used altered the pH of a 0.01 M acetate b mff the result of several trials showed less change than the error noted above. This evidence of reliability is confirmed by the work of Cady and Ingle (45), showing that the glass electrode gives constant and reproducible results for the pH during the inversion of sucrose by acid. Equal volumes were pipetted into each of the test tubes sh own in Fig. l. The volume used was enough to fill the U-tuhe to the top of of the bend ard lso fill azc1t a inch in the test tubes. This «‘3 p (I) thirty-five cubic certircters for Cell # 1 and forty for Cell # 2. The s of tli CD U-tube were then inserted nearly to the bottom of the test tubes and the apparatus clamped verticallV. Gentle suction was applied d to the tube at the top until the solutions re a.ched the top of the bend. When the apparatus was set up correctly the level in the two sides was the same, so that the liquid reached the bend from each side at the some time, and no siphoning was observable. The tee t tune scould be adjusted, if necessary, to ackieve this condition. (T E! :3 c) :1. O :5 {1) :3 Ch C‘- 3‘ o d o F4 (4" (P d p a. (1.1. a: U) C+ c: Q. 5, HQ fl 3‘ St 5 ’1 m U) r30 r+ F0 ”J G <3 0‘) :11 :3 L). W 4: U P :3 CL of the solution durirg a run, caused by elec+rolys is, never reqwti ed more than a few per cent change in the external re esis ance to maintain a con- stant volta:e between the subs id ary electrodes. After running from 150 to 250 minutes, the current was turned off and the stoyeock slowly opened, and the solution a lowed to drain back. The tale was then lifted out of file into the f.)- the liquid and a110' Had to drain as co Mp Ht ly as pore respective tes tubes. 31"» r‘v ’ (”PH *3 in "r 3'3 f n ’ .‘F ’r " 3 '1’“ "r 3 : '1 ‘ - V ‘a _ - 2 ‘ , A -‘ - 4 A. . - a I 'I .V: f J _ n ‘ A o ‘ u c ‘ , _‘ ‘ 9 ‘7‘ ‘f ”'c..f-c e.; H - t o ?”“-n-vt;1f’77 \ by - s.) -_ “ U h -‘ C‘wn .‘h I-‘.r‘ fiy-wf : 'L/“TV fi‘f 31 an '{ ‘9v-w . . er'T/‘zq n‘h l . -..a ‘..' s __u C \— ._ - L; . -1. -‘ k - 4 .1. s .4 -~u Lt; ‘ ‘ . . ~15 11ru~zr~r ij (‘9'4jv‘Ir-7 J h1~fi++fi 1v, r3, fawn 1“'I”'h‘r‘4 (I 3?“:n .- ~- .., - g. l.....uJ_ ‘ ‘ ‘4 .. . 4’ . _.~~v . I. .. _. - .9” _ -_ j 1'1 rVTrf‘ xfir'Y‘f‘ 3’ " a]_“" x A , we ‘ r“ ‘9 ”#38! V“ ~ f_md - 13:.-{Jfl t; or“ u: ore-o . -_7 e of . r' . .L c '. “‘4 . H . f‘ 3. 3 1; 1..‘t:r e 1’ 0 ‘ -+ 3:1.r" coll: Le to x, e (1 01.1 _ . '7 ’7 " 'v‘ "' ' ‘l - - . r' . I.“ ‘Q r - a J~ .- o. eflcoe, Cid t 199 ciolc c.rt;xete;s 0L corcercretei sul- ‘ ° W" A 0 .~ . u - , z .‘ +4. 3 3° I'm, - .3 : .., . - furlc 331d ( t ' -.fi-grbc} gzzude) freon-en 1.31. ‘.;€ :32. 1‘12: were duets» O”CI‘T’lII“"IT e2: Pr‘il clihjr; law! f3~wr—...Lr3 rmxreesex”r fol"fi L fi::w,11cur ‘ -‘ '~ + r :. v n -.-- + ‘7‘. r '. I. 1. 1“ n . . .1 ' ‘ . to prev ?t 931-torln;. uonplc e olilatlon 0; all C erred jiriicles vfc .+ .)4 ’1 (o :D (L 13¢?c were ellcwed to cool after hevin ." ¢ ‘ - 3. a..- ..,--f 33 3 rope of thirty pox ert hfelovxn perox dc aroiflo O 6 o 'I 0 After coolir; the sarples were diluted With about twenty CH;10 centimeters of water, and distilled in en apearatue cf th trpe deecrlbed by Allen (4:). r0 dietillate was receive boric acid, conteiuirg ten dro denser beneath the reached; the flask 'J‘) a moments lon"er. The e . \- standerd of about the rame volume mace up of a buffer of pH 5 containin l ten drops of methy necessary. The {D ;n (‘1’ 40 3 l i ’7 ”2 r i. 6 (L H. L) c+ f." ’4 H i.) (4' H. O :3 H: H 0 Pa 77 55 p) (I) of four per cent [‘4 i... 25 d‘ e ;"' ”-1 l *5 Ho <: (D O C 54 Ho 0 O C") :1 ($- t." 1 . O (-9 (D *3 U1 tip of the con- ”3 m C) " ’J 4 \D («I- J‘ 1: H *3 (D f“ U 3: i. 0 cf :3" c4- (3 urface until about 125 cubic centimeters volume was as then lowered and distillation continued for a few .itrated with N/7O acid to ietch a U) K.) [—1 r+ '49 0 9-1 3.) =3 :3 U) (+- I.) (D 73 ,+ 1 rec. The triplicates usually agreed Within 0.2 cubic centimeters of N/7O acid, or often closer. The results are recorded in Tables II and III as averages, with the "avereje deviation“ the titr tions etious. The letter i of the three titretionc frer their.t able II Mobility of egg albumin in 0.01 K acetate buffers pH Coll ff Titritians d?virt and J.-.‘b..', average cc. Left side :3 m-H .ile, O zinutes P3 18.03 £0.12 17.50 $0.10 17.67 $10.16 24.33 tC.ll 15.25 $0.35 17.63 £0.11 1". 975‘} 4.; .Uu *0.10 23.33 :1: 9.1-3 3.9.83 10.1.2 19.C5t0.05 19 .05 £0.05 y—J ;. H ") CD 156 236 '7 F“. re,“ Tallle III alburin in 0.01 M acetate huffcrs nitH flucos Titrations and average Tire, Calculated pH Cell deviations, cc. mindtes mobilities 3 Left side Right side cm./sec X 10'4 4.01 1 15.841 0.14 15.37 10.05 3-33 + 0.22 4.05 1 14.77-k0.09 14.65 10.05 181 440.153 4.15 1 15.42t0.11 15.1321036- 222 +0.065 4.31 1 13.50 $0.07 18.25 £3.10 204 +0.14?) 4.59 2 16.13t0.02 15.12.10.132 255 ——O.315 4.81 c. 15.t1310.08 16.57 £0.09 1.30 —-0.1.25 .c-2 2 16.28 £0.06 16.60-10.10 180 -0.336 5.21 1 15.43 20.06 17.18 £0.17 2.2-0- —C.422 5.2.8 1 3.22.10.08 17.25 $0.03 240 -0.577 5.53 2 16.40110. 16.99 $0.06 190 ~0.C05 6.23 2 16.63t0.10 17.18 10.12 191 -0.577 6.82 2 16.12t0.11 17.03 $0.09 190 - -O.83~O Voltage between subsidiary electrodes - 9.8 in all runs. In calculating mobilities, equatiors I and II on pages 3 and 4 were used ~- 17: m D and D: E IL— M E t I To give v in centimeters per second, the following units were used ~- E - in volts I — in amperes W - in mhos per centizeter cube in seconds d‘ I {“1{. m- .__, For the ratio m , the following expression was used -- 1 IL L~R v 2 (L+ R) 90.2 2. in which, L = average titration of left side. R = average titration of right side. V =:volume originally pipetted into each side. 13 quantity 0.2 in the denominator represents the titration of a blank. Since the voltage was always adjusted to 9.8 volts, the cor- rected value used in the calculations was 10 volts. The right electrode was always connected to the positive pole of the battery. In order to follow the convention of Abramson (5) and others, the values for the mobility are called positive when the protein is positively charged, and negative when it is negatively charse . The protein will migrate from the right to the left when it is ositively "3 charged, since the right electrode is positive; accordingly, the mobility has a positive sign when the titration in the right test tube is smaller, and is negative when the right test tube has the larger titration. The glucose used was Pfanstiehl’s C. P. grade, and all dilutions were made from one 2H stock solution, containing no measurable amount of nitrogen. The albumin used was all taken from one lot, prepared after two preliminary lots had been made. Blank nitrogen determinations using all eagents except the 3‘3 (VCR ..t 3 centimeters of N 70 acid. I-u albumin gave an avera; titration of O. o 9 o o c v v _ n ‘1' ‘ Preliminary trials us1ng pure amxonium sulfate snowed tnut 93.75 of he antonia in a na.nlr distilled cvrr. It was olso foHnd that practisilly ell cf the "ol‘tian used drained be“? int) the test +HD‘R. i‘ir‘;-five cubic C€nt‘hetor calilretr‘ test tzbrs were filltd to t}: sir? with a ; Co"°—aTPuvin soluti-n, tho sollti:n wee drawn up by suc‘iwn ard then relearol, rs previo“ly doecrih= and the twin "llcipfl +3 dr'in, Tfle d or: so in v ’ure on erch side was not perceptible, not rare tiin a few tart s f a Cqfiic n :timeter. Values for tie nvhili‘y ere ylottee in Fi;.~3 rd 0 vyered Wit. the results of others, taken fro" “Fremson (5). ‘4‘ _ 4' CAL vn II‘V \vroment of viscosi‘ . ‘n 7"”? .. 4 equation ill, “u e C, on- ,. i ' °--, has an eilect on the Phblllug of f-re, the viscosity of fl glvcose as de:cri.ed h vyv' we}, 3 used in the visc water bath at 25°. COSit V in whiz 1", 172. :- absoll‘te vi d', dz :: density of t,,‘t2 time to flo The density of M glucose was mea value obtained was 1.388. The d etate huffers used in this worn d , according to tie standard techn Five cubic centimeters each trial, and the apparatus 7 . p r J- e P r» y was determined by the equation -- scosity of liquids l and 2, re the liquids at the temperature ..L; ,- ~.‘ ith a sured w 0"! /"u ensity of water is 0.oe7 (e8) 'tphal balance at i "3 r T‘I T T I T V ——" ——l' ‘ 0—9: T, o o - .- Q o v --o-o» v1>rt -— —- u . . a n . .-o ‘- - us—f—uo . —-_ - =— ' - o - - ' o -9... _- vobob 0 on .... o o > . t ..‘t- .9 l o o ... .u—g . o . OI. . 0—0. 0 1. I o. - oooo I ’ — l u .o 1. I v 04 l..- —o- roe ‘ o — 0. o. I I _, -‘-¢.:o—o o I t o .1. ' fb - — o. : -—.— o 0-. o -o o. ——.— L Y .n-u-u..._—— --—..—— j .- A o ' on“ c *0.- . - - .n a ' 1 ‘ ...1‘ . . .1.. 'y‘ . . oo-o-' . y . o u o.o . . a. g l“ o _o —- _ ..- ool . * ~ '. . - -o a - a— no t — Q - ' o l: c.4- 3 —.. ' . I- o. -1 . — J _ 'l ' i - On ' 9". DO 0'. - L. . 3'1 0 v x . ..—. +>f§ 4—4—- 7 v No. ZOO-F. THE 8. COLE 00.. COLUMBUS. OHIO. are recorjed .h Dmnav‘q 1"” PP: v‘A -‘v 4’ &~a t o “I ,.' _r' f. ,— ~ . ..t V 4a '- s "'“"i“‘ l- I- \.'.4 L '4 ‘54»! re!" H-‘.. - , e 7. o ..L in. S n3. .3 rd w... .1 n. nQ a“. 9:1 1:. o... S 5... 1; w. nL .u, .w‘ Aw. AU fib S C .r o o O l... S 0.. Au flu do An .1 v. “I ...L ) 0 +... 0.. S C S t e n. P C e w . r t t .T. A; .1 no” “A nu U .l C. .1 n n w.” ”a .l .l 2; 7‘ h .1 r. m m .1 .H. O 1 H n; 0v r F _. e .0 S 6 C .u. a o o o f .T. F .l 7‘. A» C 9 a... an. 8 ma. V 1 I a o A .1 1 Il\ mirl .1 A.. a 1; C W. AU 2; e u o m J3 a.» nu. MU .74 KL 9.. .-l .l C -u *9 O 1x. l... .l “a S 0 CL 1i. 1:... Op. J; H. O n“ ru. a... S .9. d... ‘ ... t .4 c 2:“ a C r C V 3 .2 .T i .. . 3 h“ C no nu ._ _ .l P no .u .l C. B l 1 . C a t to r... Hm E .l a o H m-” um mm 0. 01¢ *(48) S WE‘I‘ 8 nt . \— ‘IV‘ A :‘r‘. ."~ .- V 3.";1 up.) ,, 'L. furflL-JL-.I‘ h 0 ‘7 3‘: ”E 9 m.‘«~ L 31‘). to 3 J- 95: 110;. 348 V D ,- -. v G. e viscoe ardi t:‘- Y a w ..h;\_., n '2 .43 r: ,J l “ ' 1n ' ‘1 « ~ ‘1 - \ LA \JJ..~; .._.\4& .5 - y 1 ,1 ' . I ' T -r_ + A r» 1 to understan- .to cen__ 1 ‘V‘ _. ‘ an nLFJLSUd (3, C;ns§d:r a t: electric with C? IY‘I‘ Cfif , "‘1‘ A ”CT! - centimeters. m : X : ll: t : Solving for v, 4— Lo . ~l .. l ‘1 confine }..n 71" q : when k = then VI .- ,unn.,, .J...~-~.:~A-J-wli “ : 1:. ’ L;;ffhe:.3 i' t‘ i o ' "‘ e_I, i; i_: 3 gxqi: 1t '. ' ‘2 w ‘r “ -"‘L;r-~ .3 T? an. , ,- . tluns for V4 3» e;‘.et ‘s I &“u ll, ,-_ee 3 an” “l"??"f" derivation is reen‘icfrjg’ tilt of Fair-.3 ( O C ‘v 0 O 1«“2 filled with a c: loii 1 Vol, currvinf an r A; +.'. + q— -.: P ~- a cr-ss -s,1on at I'e To nt C; q elders ‘0' . ‘\\'I 'V )'-’-V ‘v H v ‘.~ “"\ 'r’s53rif roll..3d tits ;€e;: w ITLO;€)'t.C c;,;r2 O O 0 section Q in time t f . . -‘ ,_ . .L‘ ., f old st“ ngeh at cross section q O 9*. .fi #. . m\hilitj o: p"rticles ° . .1 0 V , r. 6‘ - time for union c.rrrnt Lists 0 O O O 0 v- _‘ : original concentration of sol, in n::s gel Lust I u 0 volume, using fwe sine rth :8 far 3. r f ’4 X (1 t k X 0 nrrcnt flpUlW; _ - v t, .1..- ';- p w ., 4. sFBCLfic vtxd cthLaj or con unto: .4! ‘3 (Q’- HI (I ,3 h I .1 (.4. 0 fl ”1 q c+ (P T (-9- V n‘.’ 4". .r‘ ‘3 o. 1"". -‘.- henry and wiltufiln (a) nozlvei toelr delf- [‘1 _ .L . 4.... ‘V . .. ‘ ' - 2 ..V01u3;8 between bio fined elgotrtces 1n the sol 9_ ' F 3 .n., _’ -, >,\. 4- WLlCh is C¢TFYLLQ the CUCTKJL. A w” . ’- \" v q' . ‘ ? w . 1", D :=1 constant dopenting unlj on too position of toe ‘3’ electrodes between elich o is measured. Therefore - In all these equations, the proper units must be used; C. G. 3. units were used in calculations. However, instead of using M and m in actual weights of protein for the calculations, the ratio m/K was determined in U) terms of the titrations. ince the percentage of nitrogen in the albumin was constant and the same concentration of acid used to titrate all samples, the titrations were in a fixed ratio to the corresponding amounts of protein. The gain on one side must equal the loss on the other, so a number proportional to M can be obtained by averaging the tit-etions of the two sides. The titration of a blank must be subtracted from h average, however. This gives for a number prOportional to M, 1.1rrR.) -0 2 as used on page 22. Correspondingly, the amount of protein transferred was proportional to the change in concentration of each side multiplied by the volume 0" each side, or (IP‘R) These are used to calculate 2 O the mobilities as on page 22. It will be seen that to use the equatiors above the follow;rg conditions must be met -- .J t t- J ‘3 1 'I I. D i- I. I V? , i 0 ll 5 ) I... 0 Q ‘5 “.1. I I I ) .1 ‘ D .1: J 6 '7“ - ,.~'v.+—°. .— 1.41,? t. .-.L 3 ,W‘. -.W - '- . u. lie anum -isel :2. e- N.Ie o;tci lne nu;; tne amount of .~ _‘l' - w.” ..71,! 72-.-- A , .7 we “4.“ “a 1 Tie turr- n- ~ +. w - cm): «a .H , 7. ,c , ~.sr‘t urnstant mu. . J -5u \IJ_..~-“'-' . . 1 e 1 . - ., -. ~ ’1 ' I. - 2 J t I ,‘le- ‘ Ier (h vr 51’. P 7 car" a .- |-‘ ‘1- ' (-_. a, . _ ., A _‘__,,,_a "“‘J --ul .--1 '0. '; ...9 V” I.“ .1 .. v~ao r- ?" ._ (hash: 2.1 (...p ““4. 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P'L“ \v "r-- w~~~L~ r.-.-1\ - "‘ PM ME l“;f“'\ 2. Tue to “be la r“ “or tr Lite, .Lon m;_e ;o tern t.; ,-.1 - o c, u n 3. CE; gee 11 co.3ocltlt" c: t 3 1L1 ii ‘3 ' n *“e €33- . .‘ '- t "V v v'w’..-y"', m‘ .r‘ : .1 *‘ Q . " ‘ 51 Lery Ll- " r3 3: mist be ovo_-;3. -313 llm1ts 1 t t1 0 for "‘1PH tte .- 4» -.~ ‘ 3. A _- ,. 3.. . -.- 3 ,. - . .31 current any be a;lcxfa to flql, 333 consellartlv L13 are.ricg _f t 3 method, Sane only small changes in C).c;3tr3tion esult. q o a o 4. Tue “ethcd dxes qu 33 fl 1-231- r 1‘11: *3 +_r“3:€t re control n .‘a3 an ‘ y‘v‘ - 7. ”0' -;.\~ -. P34 "'1“ L .- ‘-. ,‘L. {if} ,:gyr‘w-J .- . (u -: - +‘-~o 3-..e b-I._ct eALl)- r. ' _._ 33).; J) ~J.L or": Q \14-3'J... .-ZzL/Tan‘-.‘.« .11; .Liu 3 L . ,_.. 2. . ‘ .. ' 1' -‘f" .WLP . V"~«.-‘ .— ,.,, h '. ' present uorl 13s due to 3331311331 l1-f13t;_1es. so; instgos', 1f tne .3 . ' ‘. "D h "' '2 ’ "" 'P 1'“ fij’ "‘-\: ~"“.--r+-n‘/I 9? tltrfialon ‘xJL echtl CSJJJC 'lll 81~e 1.1:.131‘13111T‘z; *UOUJ (ft-’EJJC CSII-‘vl‘ :3. "1‘; ’ L“. 3 '.- P .‘31 43*. -J r... ‘- ‘.~ ,....._6. ..-‘ - trial in w ion has Curfrnt flczeo 1o1 th.ee TJUFS, toe ctlieslcndln; a I ,4 :1? ‘. y . . ‘ - o r . wv- -4 r ‘.- “ ‘ \ '-"* - uncezi M1 :ty in tue mobil1gg would be ebont‘:0.13 a 13 centiretcrs ,cr 1 second. T51: sue ~sts that the :ood arrcotont of tr.e d:L ta for e33 6+ 0 tr .1 albumin el3r e "ith the work of others must be conccaeu .3rtly I . fortuitous; on the oth3r h3nd, sin ce e“rors of this rind ieyend purely ‘V on chance, it s Wozld he possible by sufficient repetition to rednce the errors considerably. There was no attempt made to control the temperature of the V liquid in tlie cell. However, the 3H3 ll curror nt whic n flowed coul i not maintain more than a sli3ht tempera. ture difference between the cell and the surrounding air. Paine (8) showed with a cell similar to the one used in this work ILat the kufl'available from the CUTPCTLt flot irj won A not maintain a difference of more t.han O. 20 betwe en the cell and the air around it. The variation in room toofiera+or would be more im portant thm this, but it would stillr ot cause on error comoorable to the ana alytical uncertainties. The largest uncertainty in meas iring the Volta :e was that due to the effect assumed to be folerization. Any error fron t! is source would therefore be practically constant. There ntst be considsred the effect of changes in corrosition produced at the main electrodes. This would not affect the determination until the change had had time to migrate to the region between the sub- sidiary electrodes, where the mobility was being determined. The mobility of the hydrogen ion is much the greatest of all ions, so it will not be necessary to consider any others in this relation. The mobility of hydrogen ions is about ten centimeters an hour in inorganic electrolytes; , the presence of colloidal particles would tend to decrease it. The field strength in these experiments was never greater than one volt per centi- meter, and the distance from each main electrode to the nearer subsidiary electrode was about twenty centimeters. Therefore, no disturbance at the main electrode could reach the subsidiary electrode in less than two hours. It would require some time for the buffered solution at the main electrode to change in pH, with only a small current flowing, so no important changes could take place for a still longer time. This indicates that the time of running was not too long, and the results with egg albumin alone bear out this contention. The concentration of egg albumin was not kept constant in this work. A slight error might result from this, since Smith (18) has shown that the pH of zero mobility of egg albumin, as determined by the micro- scopic method, decreases slightly as the concentration of protein increases; but the change in mobility due to this effect would not be large enough to be appreciable in this work. These numerous sources of error should be investigated further in a more careful study of this problem; but the foregoing considerations dispose of them satisfactorily for this work. 31 The fact that egg albumin, in the absence of any other compounds except acids or bases, will increase in mobility as the pH is changed farther from its isoelectric point, is obviously related to the develop- ment of charges on the albumin particle by reaction with acids or bases. Abramson (3), by application of modern ionic theories, has deduced the prediction that the mobility of a protein at any pH should be proportional to the amount of acid or base bound at the same pH. According to this, an increase in mobility is due either to an increased dissociation of groups reacting with hydrogen or hydroxyl ions, or to the dissociation of new groups. ‘5‘ _Il ‘3-dSF-L' ;F:i§imm‘i If the equation relating mobility to the zeta potential is considered .. g D X E 4 7T 9 v1? it would seem that changes in mobility might be predicted from changes in D and , provided other quantities remain the same. This was done by Daniel (26) for the effect of alcohol on mobility. It was necessary in her experiments to make allowance for the change in titration curves which alcohol caused, since as explained by Abramson (3) this would affect the zeta potential. When this change was taken into consideration, the change in mobility was that predicted by the change in viscosity and dielectric constant of the medium. If it is assumed that glucose does not affect the titration curve of egg albumin, the same comparison can be made between the different results in this work. The dielectric con- stant given in the International Critical Tables (48) for molar glucose, which is 16.92 by weight glucose, is 59.5 (obtained by interpolation). The effect of small concentrations of salts may be disregarded as has been generally done in calculations of this sort. The viscosity of the glucose solution was 14.5 millipoises, and did not change appreciably on 32 the addition of buffer. If in the absence of glucose the dielectric con- stant is assumed to be 81.9 and the viscosity 8.8 millipoises (the values for pure water, which are not changed greatly by the concentrations of buffer used), the ratio of the mobility in molar glucose to the mo- bility in the absence of glucose should be 0.44 to 1. Examination of the curve for mobility in glucose solution shows that this is approximately true in solutions more acid than the isoelectric pH, but farther from it in the pH range 4.5 - 6.5. In fact, the mobility at pH 5.2 is almost identical in the two solutions. While the mobility curve may be in error as much as 0.10 s:10dye¢ any point, it does not seem that experimental errors could account for a larger effect. Consequently these results 11—1.,“ W. “as A indicate that in the pH range 4.5 ~ 6.5, the glucose might affect the albumin in one of the following ways - 1. By changing the zeta potential. 2. By changing the acid or base bound. 3. By changing the dielectric constant in the double layer. 4. By changing the thickness of the double layer. - or in a combination of these, or by some other effect not yet understood. The further observation can be made, that above pH 5.2, the mobility in molar glucose remains practically constant, within the errors of the method, or at least it increases much more slowly than at any other pH shown. This could be caused by all conditions remaining con- stant, including the amount of base bound, which is contrary to the usual behavior of a protein as the pH is changed, or by a change in one or more of the factors enumerated above sufficient to compensate for an increased binding of base. Whether any one of these causes is more probable than the others cannot be proved on the basis of present knowledge. These effects may be related to the findings of Gubarev and Moiseenko (41) that the amount of glucose adsorbed by egg albumin in- creased in the pH range 4 ~ 8, as the pH increased. They may also be connected with the other effects reported, the decrease in reducing power and sweetness of glucose when egg albumin was added. A more remote con- nection may exist with the effects of polysaccharides on various amino acids, peptides and proteins, discovered by Mystkowski, Rafalowska and others. The results suggest a number of ways of further studying similar problems. Mobilities might be determined over a.wide range of concentrations of sugar, with different sugars and with related compounds. The titration curve of egg albumin in sugar solutions might be examined, to test the possible variation referred to above. The rate of denatn uration at different acidities in sugar solutions might also yield information. The protective effect shown by Duddles (2) might vary with the pH; his experiments were done at pH 4.8 and 5.2, both in the range in which glucose seems to affect the egg albumin. Below the isoelectric point different results might be obtained. The result Duddles obtained in saturated glucose solution is of interest in connection with the ultrafiltration experiments of Gubarev and Moiseenko (41). Duddles heated egg albumin in saturated glucose, then dialyzed it to remove the sugar, and adjusted the pH to 4.8 . Since glucose does not all pass through an ultrafiltration membrane when egg albumin is present, it would not be expected to pass through a dialyzing membrane; so probably there was still sugar present after dialysis, which might help explain the clear- ness of the solution when the pH was adjusted to 4.8. In this work surface denaturation was noticeably less in the presence of glucose than in its absence. 34 CONCLUSIONS AND SUMKARY 1. Egg albumin was prepared by a slight modification of the method of Keckwich and Cannan (44), using sodium sulfate to precipitate the albumin. 2. The mobility of the egg albumin was determined in 0.01 M acetate buffers, over the pH range 4.0 - 6.5, with and without the addition of glucose to one molar concentration. 3. The viscosity of one molar glucose was found to be practi- cally unchanged by 0.01 M acetate buffers. 4. In solutions more acid than the isoelectric point, the effect of glucose on the mobility of egg albumin was, within the exper- imental error, that which would be expected from changes in the viscosity and dielectric constant of the medium caused by the addition of glucose. 5. In solutions in the approximate pH range 4.5 a 5.2, the mobility is larger than would be expected; it is nearly the same as in the absence of glucose. 6. In the pH range 5.2 - 6.2, there is little change in the , mobility when glucose is present in molar concentration. The increase is less than would be expected from comparison with the results for egg albumin alone. .-.l “*yurr..a ... r -..-- ‘1.-. ' 'T". , ’ A .-. 1' ‘ Ox 8,; ”l1 '14. AHSSLS ,5; z 3 n ‘ 1 W fi ’l .3 n" 7 f‘": r. . 1‘ Fc‘. 1‘ W {‘1 '5‘ CU...L~F., '. ’ .,.~L._ t .‘Jgi.’.,‘. _r.v, . ”Lil. ...Ju'e... . -4. ' q' .,. 1. v I l. ' "f‘ ‘1' “‘-o— VUL‘J.§AL-V11Lu -V I. '~. Mtl‘f-l' ON", 2* A0, *9. w-w’r-mu—P. --e hi0ChC“i"f”f 0rd rsiicine. The raw vgv, (Wu *\ O'»; .’ as .s ...ue,‘:., TcEain, J. 3., aid T,V‘cn, 3. 3., T‘s Ti rs‘llr Hgfi f-r ‘:La“"izr ni""i field "’*ratirn, electronh~r= .; - - ‘-‘ ’J "J r O O and ianzvstion of corpourds of ,2 I)’- ‘ 7 fin aflq.‘ fret“ . n . C .. ' ’x A ..OlCI. , O 5") , (L-*‘;:I{S- 3.9.1, .,, 1" an e1, L., and Pauli, u., uie :estisu He :3 keit you Vulloidionefi 126 : 247, (1927) Paine, H. M., 28) (f Soc., 24 : 412, (1 (I) u-Il' Cataphoresis in condor oxide . ,\~ ~:+ w-+s hWPvdhe an: 4 A ‘ .7 .7 ~ v- \ e-.J;_; ~r~\ #4 V~UI .. h.-- ‘7 pv 4‘ . “fih'w‘1r‘+~ ». J. -_ - . K - .4. Ja- ‘ JA..~.4.L H Cr ‘- a- v e '3 : P‘fl (‘4 —-t.3 A. o ‘1’, . _ \ .-v.) M can u- 3 . (‘1‘- . N} v :v ("'"r "\ A" ' ‘ "” --_ Lulk’.a LIL "am ’- a i.” .. V O . m; 'f‘“(. - it 1., wfifltM—L‘}; J x k~ .. r. s 4.“,14‘ _‘ «en‘s» - a. O '7 F~..-..,° 1, [J .1. .LL. fly“ _- J » JLACLQ, 10. ll. 13. 14. 15. 16. 17. 18. 19. Y" '4 Henry, D. 8., and Erittain, J., Cataphoresis. art III, Comparison of the transport and moving boundary methods. Trans. Farad. 306., 29 : 793, (1933) Svedberg, T. ard Jette, E. 3., The cataphoresis of proteins. J. Am. Chem. 323., 45 : 954, (1923). Svedbere, T., and Tiselius, A., Determination of the mobility of proteins. J. Am. Chem. Soc., 43 z 22 2, (1926) Bi Tiselius, A., ~e moving bourdary method of studying the electro- phoresis of proteins. Dissertation, Upsala, (1930) Fattscn, 8., The relation between flocculation, adsorption, and the charge on a particle, with especial regard to the hydroxyl ion. Kolloidchem. Beih., 14 : 22 , (1922 Northrup, J. H., and Kunitz, M., An improved type of microscopic electrocataphoresis cell. 3. Gen. Physiol., 7 z 729, (1925) Abramson, H. A., Modification of the Northrop-Kunitz microcataphoresis cell. J. Gen. 2239101., 12 z 469, (1929) {ardy, fl. 8., On the coagulation of proteid by electricity. J. Physiol., 24 : 283,(1899) Loeb, J., Proteins and the theory of colloidal behavior. meGraw- Hill Book Co., Inc., New York (192 ) Smith, E. R., The effect of variations in ionic strergth on the apparent isoelectric point of of; albumin. J. Biol. Chem., 108 : 187, (1935) Smith, S. R., The influence of the method of preparation and of cations on the isoelectr1c point of ovalbumin, J. Liol. C 22., 113 z 473, (1936) SO. '17 .54). m .43 e '- ie T) . F-J , .° 1" :1 '7“ Bl l‘- a, All. A-.’ AIL“: ‘ r1»,- e1n(\* ‘\r~(‘7' \rdA‘d .- -~. .- ' \~‘- 7‘ fl (‘1‘ ,‘nr "' . . ' 1 r: ’ a. o (v.1- U. \ o, - u C C"'.‘!'."+‘ «‘1 P7“ fir '—-~‘-.-. a - J- h n7 ~~,7w\ uL’ --ui, D ‘ V'.. ‘ 54‘ V, .’ . A. J j'-.v1.§01nc Db" \1K 5 ~U.L a .' l A. A 'y.. TH “I- v -‘ r‘ w.) ,: ~ "01 -‘ -.‘J _’ A -..o “r: —~ r. "2 "1' a . ‘uy , a. ., s .4 -17.."' cu -'. - (PF‘ - A ' . 7 'IN $" ' '1 I + 1 ‘ mm! J L, o ., Jami-.2! 1- ..h..,\ '- t" , ._. .8 C . a- h D? a: _ w 1 A . ,. 1 U 4. . -.’ a "r.. . 1 - ll rjjwr‘ . ~ w...., L . 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