VISCOSITY OF SOLUTIONS OF DENATURED AND OF NATIVE EGG ALBUMIN

BY HENRY B. BULL

(From the Department of Chemistry, Northwestern University Medical School, Chicago)

(Received for publication, Jurle 26, 1939)

There have been several equations derived which express the relation between the viscosity of a suspension and the degree of asymmetry of the particles. Among these equations may be mentioned that of Kuhn (7) which has been employed by Lauffer (S), Neurath and Frampton (lo), ru’eurath and Saum (II), and Neurath (9) to estimate the ratio of the length to the diameter of the protein molecule. These studies have been most interesting and it is largely due to their stimulation that the present re- search was done.

Recently, Polson (13) has formulated an empirical equation re- lating the asymmet.ry of protein molecules to the viscosity of their solutions. At the present stage of our knowledge of the compli- cated relation between asymmetry and viscosity, an empirical equation such as Poison’s seems preferable to one derived on a t,heoretical basis involving more arbitrary assumptions.

A search of the literature reveals that the viscosity of solutions of native, of heat-denatured, and of urea-denatured egg albumin has been determined by various investigators. The data reported are, however, not suitable for the present calculations, since they were in general determined at higher protein concentrations at which the viscosity to protein concentration relation departs from a linear course. This point is particularly important in the case of the denatured protein. Some of the measurements have also been made without regard to the electroviscous effect and there has been a curious absence of description of the apparatus and experimental conditions under which the viscosity measurements were obtained. The careful and exact technique of Jones and

39

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40 Viscosity of Egg Albumin Solutions

coworkers on the viscosity of electrolyte solutions demonstrates that the determination of viscosity by the use of the Ostwald viscosimeter presents many difficulties which must be resolved and it seems evident t,hat the experimental conditions for viscosity measurements of protein solutions should be rigorously defined and described.

EXPERIMENTAL

Egg albumin was prepared from fresh hen’s eggs by the method of Kekwick and Cannan (6). The albumin was recrystallized four times and dialyzed against running distilled water until sulfate-free. The various preparations were between 97.5 and 99.0 per cent heat-denaturable. All preparations were water- clear.

The viscosimeter was of the Ostwald type and made of Pyrex glass. The drainage bulb was cylindrical in shape over nearly its whole length. The reservoir “bulb” was a cylinder of uniform diameter over its entire length with a 45” cone connecting it to the transverse tube which led to the capillary.

The viscosimeter had the following dimensions and charac- teristics: diameter of reservoir tube (inside) 1.65 cm., diameter of drainage bulb (inside) 1.00 cm., diameter of capillary (estimated from rate of flow) 0.0592 cm., length of capillary 20.1 cm., volume of drainage bulb 3.91 cc., usual working volume 10.0 cc., time of outflow of water at 35” (with 10 cc.) 98.75 seconds, average pres- sure head (with 10 cc.) 1.92 X lo4 dynes per sq.cm.

The viscosimeter was clamped in a rigid metal frame and never removed from it. Dichromate cleaning solution was allowed to stand in it when it was not in use. Before a measurement it was washed repeatedly with water and dried by sucking air through it. After a series of readings were made on a solution, it was washed out wit’h hot NasP04 solution and then with water and dried. This treatment was found to restore the water con- stant. The water constant was determined each day and was found in the course of 9 months to have dropped gradually from 99.07 seconds at the beginning to 98.75 seconds at the end. The solutions were placed in the viscosimeter wit’h a standardized pipette.

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H. B. Bull

The experiments were carried out at 35’ Z+Z 0.01’ in a well stirred water bath. It was found to be most important to have adequate stirring. The time of outflow was determined with a 10 second dial stop-watch a.nd the time could be estimated to 0.01 second. The accuracy of timing was greatly improved by the following technique. Two 0.5 cm. lengths of rubber tubing were placed around the glass tube above the drainage bulb, as well as around the capillary below the drainage bulb, in such a fashion as to expose the calibration marks as narrow slits. A strong beam of light was allowed to strike the viscosimeter at right angles to the observer. Under these conditions the glass tubes at the calibration marks when filled with liquid reflected no light, but when empty appeared brilliantly illuminated. It was frequently possible under these circumstances to achieve a series of outflow timings which did not differ more than 0.01 second from one another.

It was found important never to allow the protein solution to dry in the capillary between measurements, but to draw the solu- tion back into the drainage bulb immediately after an observa- tion had been made; otherwise air bubbles and bits of coagulated protein would cling to the capillary. If the solution was drawn back into the drainage bulb immediately after a measurement, the small amount of surface-coagulated protein which always formed would be carried into the tube above the drainage bulb and would deposit there as the meniscus dropped on a subsequent measure- ment. In spite of these precautions, however, air bubbles and threads of denatured protein occasionally formed in the capillary and these were removed with a drawn-out glass rod manipulated through the upper tube of the drainage bulb. The tendency to form air bubbles was greatly decreased by boiling the distilled water which was used to make up the diluted protein solutions.

It was established that the viscosity of protein solutions as measured by this viscosimeter was independent of rate of flow, by using working volumes of 5, 10, 15, 20, and 25 cc. Since the reservoir tube was a cylinder of uniform diameter, increasing the working volume by 5 cc. amounted to decreasing the average pressure head by 2.34 cm. of water. The constancy of the vis- cosity of a 0.2 per cent solution of heat-denatured protein at pH

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42 Viscosity of Egg Albumin Solutions

9.44 with varying working volume is shown by the following figures.

Working volume cc.

5.0 10.0 15.0 20.0 25.0

t/to

1.0237 1.0248 1.0236 1.0236 1.0234

Since the viscosity is independent of rate of flow, it was con- sidered unnecessary to apply a kinetic correction, as a kinetic correction should vary with rate of flow. The figures above also indicate the absence of &ructural viscosity. This experiment was repeated at pH 4.19 and at pH 2.50 and both showed constant viscosity with varying rate of flow.

The specific viscosity (specific viscosity is equal to the relative viscosity minus 1) was studied as a function of protein concen- tration. To our amazement the specific viscosity to protein concentration curve showed a hiatus. It was found that when actual times of outflow were plotted against protein concentra- tion the curve when extrapolated to zero concentration inter- sected the time axis at a point greater than the water constant of the viscosimeter, in spite of corrections having been made for the viscosity of electrolytes present (see Fig. 1). The difference between the time of outflow of the electrolyte solution and that found by extrapolation of the concentration-time curve, varied with pH, being, in general, lower in the basic region than in the acid and disappearing in the extreme acid and basic regions. At pH 2.60 the difference in times of outflow was 0.50 second and at the isoelectric point it was 0.22 second. Away from the isoelec- tric point the hiatus in the specific viscosity-concentration curve did not come until concentrations higher than 0.3 per cent of protein had been reached and, in many cases, especially on the acid side of the isoelectric point, t,he hiatus was not realized even at 0.8 per cent, which was the highest protein concentration used. The extrapolated time of outflow of both heat- and urea- denatured protein solutions was always normal and equal to that of the water constant of the viscosimet,er when corrected for the presence of electrolytes.

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H. B. Bull 43

Much time and effort were spent to ascertain the cause of the extraordinary hiatus in the viscosity to protein concentration relation. To this end the following experiment was done. The clean viscosimeter was filled with protein solution and allowed to stand for a few minutes with the solution in it. The solution was then withdrawn and the instrument washed thoroughly with water and finally dried. It was found that after this treatment the water constant had been increased by 1.00 second. The in-

99.ob 0.1 0.2 0.3 0.4 0:5.6

Protein concentration in per cent

FIG. 1. Time of outflow of egg albumin solutions at the isoelectric point as a function of protein concentration.

strument was then thoroughly cleaned with Na3P04 solution, washed with water, and dried. Protein solution was then placed in the reservoir tube but not allowed to enter the capillary. After standing, the protein solution was withdrawn and the instrument washed with water and dried. The water constant under these conditions was 0.92 second above that of the clean instrument. This indicated that conditions produced by the presence of protein in the capillary had little if anything to do with the hiatus in the viscosity with increasing concentration. It was noticed that after the inside of the viscosimeter had been coated with protein by

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44 Viscosity of Egg Albumin Solutions

allowing a protein solution to stand in it, which was then washed out, the water draining from the drainage bulb made a 0 angle of contact with the glass surface, while the ascending meniscus in the reservoir tube made a 90” contact angle. With protein solu- tions the descending meniscus always made a contact angle of 0 with the surface of the drainage bulb (surface completely wet). The ascending meniscus in the reservoir tube made angles of contact less than 90” but only with higher protein concentrations was complete wetting achieved. This behavior suggested an ex- planation based upon surface tension effects.

Jones and Fornwalt (5) have considered the corrections which are necessary when one is dealing with a liquid whose surface ten- sion is lower than that of water. Here the situation is somewhat different. The surface in the drainage bulb is being continually destroyed and created as the protein surface is deposited on the interior of the drainage bulb as the meniscus descends. Under these circumstances the surface tension must be high and fairly close to that of water. In the reservoir tube, however, the sur- face is not destroyed and upon aging surface tension is probably in the neighborhood of 46 dynes per cm. (unpublished experiments by the author on the surface tension of egg albumin solutions). In addition, as has already been pointed out, the ascending res- ervoir meniscus does not completely wet the glass surface, so that the surface tension is not completely effective in tending to draw the liquid up in the reservoir tube.

The following simple calculation has been carried out for the case of pure water in the viscosimeter which had been previously coated with protein and whose ascending meniscus in the reservoir tube made a 90” angle of contact with the glass surface; from Poiseuille’s law of liquid flow in a capillary we have

where VI is the volume of liquid flowing in time tl, a is the radius of the capillary, X is the length of the capillary, 77 is the viscosity of the liquid and P is the pressure forcing the liquid through the capillary. P, the average pressure head, was calculated in the following manner. The time of outflow of pure water in the clean viscosimeter with a 10 cc. working volume was determined. The

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H. B. Bull 45

working volume was then increased by 5 cc. and the new time of outflow obtained. The addition of 5 cc. of water was equivalent to decreasing the average pressure head by 2.34 cm. of water. Two simultaneous Poiseuille equations were obtained. By elim- inating the constants common to the two equations, an expres- sion was obtained for the average pressure head in terms of the two times of outflow and the decreased pressure head due to the addition of the 5 cc. of water. The average pressure for a 10 cc. working volume was calculated to be 1.92 X lo4 dynes per sq.cm. a, the average radius of the capillary, was calculated by substitut- ing the average pressure head and the other known quantities in Poiseuille’s equation and solving for a.

For a liquid making a contact angle of 90” with the reservoir tube, however, P has been decreased by the pull exerted by t’he surface tension of pure water on t,he wall of the reservoir tube, so that we have in this case

where g is the surface tension of pure water at 35” and assigned the value of 70.7 dynes per cm. and r is the radius of the reservoir tube and is equal to 0.825 cm. Equating volume VI to volume Vz, and transposing, we have

t1 -=1-2” tn Pr

Substituting numerical values, we find that tl/tz is equal to 0.9919, or, since tl is equal to 98.75 seconds, ts is equal to 99.65 seconds and the difference between the water constant of the clean vis- cosimeter and that whose interior surface is coated with adsorbed protein is 0.90 second, which compares favorably with the ob- served difference of 0.92 second. This calculation shows that surface tension effects are entirely capable of accounting for the increased water constant of the viscosimeter. The hiatus in the viscosity-concentration curve is probably due to abrupt changes in the protein surface film as the protein concentration is in- creased. Bull (1) has already described such changes taking place at about 0.2 per cent egg albumin concentration. Such changes would be expected to alter the wetting qualities of such a film.

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46 Viscosity of Egg Albumin Solutions

Results

Fig. 2 shows the specific viscosity as a function of protein con- centration for solutions of native, of heat-denatured, and of urea- denatured egg albumin in the presence of 0.2 M phosphate buffer at pH 8.00. The heat-denatured solutions were made by adjust- ing the protein solutions to pH 8.00 with 0.1 M NaOH, heating on a boiling water bath for 7 minutes, cooling, and adding the phos- phate buffer and making up to volume. The solutions of urea- denatured protein were prepared by adding 1 gm. of urea per cc.

0.10

0.06

%

0.04

0.00 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Protein concentration in per cent

FIG. 2. The specific viscosity of egg albumin solutions as a function of protein concentration. Curve 1, urea-denatured egg albumin; Curve 2, heat-denatured; Curve 3, native egg albumin. All solutions are at pH 8.0 in the presence of 0.02 M phosphate buffer.

of the protein solution. At the end of 1 hour’s standing at room t.emperature the phosphate buffer was added and the solutions made up to volume and their viscosities determined. It will be noted that the viscosities calculated for the 0.2 and 0.4 per cent native protein solutions are slightly above the straight line con- necting the values for the 0.6 and 0.8 per cent solutions with the origin (see Fig. 2). This is the same effect as is shown in Fig. 1. The specific viscosity of the heat-denatured and of the urea- denatured solutions did not show this effect and all points for the denatured protein solutions fall on a smooth curve. It is also to

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H. B. Bull 47

be noted that the viscosities of the solutions of both the urea- denatured and heat-denatured departed from a straight line rela- tion with increasing protein concentration.

The question arose as to what extent t,he protein in contact with the urea had been denatured. To determine this 1 gm. of urea was added to each cc. of protein solution at the isoelectric point, and the solution allowed to stand at room temperature for 1 hour. The solution was diluted and brought back to the iso- electric point with the addition of acid (there is a change of pH upon denaturation), a small amount of Na2S04 was added, and the precipitated denatured protein centrifuged and washed twice with water by decantation. The precipitate of denatured pro- tein was finally transferred to a weighing bottle and dried at 105” to a constant weight. That all the urea-denatured protein had been precipitated was established by the fact that the filtrate gave a negative nitroprusside test,. This analysis showed that 86.5 per cent of the protein had been denatured by the urea treatment. The viscosities of the solution of urea-denatured protein, as is shown in Fig. 2, really represent, therefore, the viscosity of mix- tures of 86.5 per cent urea-denatured and 13.5 per cent native protein. Estimates of the specific viscosity of solutions of com- pletely urea-denatured protein were made by calculating the time of outflow of a native protein solution whose concentration was 13.5 per cent of the total protein concentration. This time of outflow was then used in place of the water constant from which the relative viscosity of the urea-denatured protein solutions, whose concentrat.ions were 86.5 per cent of that of the total pro- tein in solution, were obtained. This calculation assumes the viscosities of the urea-denatured and native protein solutions to be additive.

The volume of protein per cc. of solution was then calculated by multiplying the weight of prot,ein per cc. of solution by 0.746 (specific volume of egg albumin). The values obtained were di- vided into the corresponding specific viscosities and this function (le/p) plotted against’ protein concentration. These curves were extrapolated to 0 protein concentration to yield values for vs/cp at 0 protein concentration. The vs/cp values obtained in this manner were 5.5 for native, 9.3 for heat-denat’ured, and 12.3 for completely urea-denatured protein.

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48 Viscosity of Egg Albumin Solutions

An attempt was made to determine the viscosity of egg albumin denatured by guanidine hydrochloride but it was not found possi- ble to prepare solutions of protein so denatured that were clear and did not contain visibly suspended particles.

DISCUSSION

The Polson equation (13) can be written in the following form,

qs,(o = 4.0 + 0.098 (a/b)2

where a/b is the ratio of the major and minor axis of a prolate ellipsoid. Substituting the gs/(p values reported above, we have for the native egg albumin molecule an asymmetry of 3.9 : 1, for heat-denatured 7.4: 1, and for urea-denatured 9.2: 1, all at pH 8.00 in 0.02 M phosphate buffer.

It is evident from these studies on viscosity that even urea-de- natured egg albumin still has considerable structure and is not simply a polypeptide chain in the p-keratin form, since the egg albumin molecule, if it existed as a P-keratin chain, should be about 1000 A. units long and about 10 8. wide, which would yield an asymmetry of 100: 1.

It is interesting to note that Greenstein (3), in a study of the sulfhydryl groups exposed, upon denaturation found 0.5 per cent sulfhydryl expressed as cysteine for heat-denatured and 1.06 per cent for urea-denatured albumin. The greater sulfhydryl for urea denaturation parallels the greater asymmetry of this form of the molecule.

Williams and Watson (14) reported the molecular weight of urea-denatured egg albumin to be 21,000 as determined by the ultracentrifuge when t’he diffusion constant of a spherical particle of this size was used in the calculation of the molecular weight. This finding is not substantiated by osmotic pressure measure- ments of Huang and Wu (4) and by Burk and Greenberg (2). It is probable that the apparent discrepancy between the value obtained by the ultracentrifuge and that by osmotic pressure measurements may be due to the fact that the urea-denatured egg albumin molecule is not spherical but has, as shown by this re- search, an approximate asymmetry of 9.2: 1 as calculated by Pol- son’s equation. Perrin’s (12) equation relates the asymmetry of a molecule to the ratio of the real diffusion constant to that of a

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H. B. Bull 49

molecule having the same mass but spherical in shape. Sub- stitut,ing the asymmetry of the completely urea-denatured mole- cule as determined by Poison’s equation in Perrin’s equation, WC find that t.he real diffusion constant of the urea-denatured egg albumin is only 0.66 of that of a spherical molecule of the same weight. Substituting this value in the sedimentation equation and using Williams and Watson’s sedimentation constant, we find the molecular weight of urea-denatured egg albumin to be 32,000. This value compares favorably with that reported by Huang and Wu of 32,500 and lends support to the degree of asymmet,ry as calculated by Polson’s equation.

SUMMARY

1. The technique is described for the measurement of the vis- cosity of dilute solutions (less than 1 per cent) of egg albumin.

2. A remarkable hiatus in the concentration to apparent vis- cosity relation is described. The cause of this hiatus is suggested as being due to lack of wetting of the glass surface by an advanc- ing surface of dilute egg albumin solutions. As the concentration is increased, the wetting characteristics of the surface film abruptly change. The more concentrated solutions wet glass completely.

3. The viscosities of solutions of native, of heat-denatured, and of urea-denatured egg albumin have been measured and the asymmetry of these forms of the egg albumin molecule calculated.

BIBLIOGRAPHY

1. Bull, H. B., J. Biol. Chem., 123, 17 (1938). 2. Burk, N. F., and Greenberg, D. M., J. Biol. Chem., 87, 197 (1930). 3. Greenstein, J. P., J. Bid. Chem., 126, 501 (1938). 4. Huang, T., and Wu, H., Chinese J. Physiol., 4, 221 (1930). 5. Jones, G., and Fornwalt, H. J., J. Am. Chem. Xoc., 60, 1683 (1938). 6. Kekwick, R. A., and Cannan, R. K., Biochem. J., 30, 227 (1936). 7. Kuhn, W., 2. physik. Chem., ilbt. A, 161, 1 (1932). 8. Lauffer, M. A., J. Biol. Chem., 126, 443 (1938). 9. Neurath, H., in Cold Spring Harbor symposia on quantitative biology,

Cold Spring Harbor, 6, 196 (1938). 10. Neurath, I-I., and Prampton, V. L., Science, 87, 468 (1938). II. Neurath, H., and Saum, A. M., J. Biol. Chem., 128, 347 (1939). 12. Perrin, F., J. physiq. et radium, 7, 1 (1936). 13. Polson, A., Kolloid Z., 88, 51 (1939). 14. Williams, J. W., and Watson, C. C., Nature, 139, 506 (1937).

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Henry B. BullALBUMIN

DENATURED AND OF NATIVE EGG VISCOSITY OF SOLUTIONS OF

1940, 133:39-49.J. Biol. Chem. 

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