THE DENATURATION OF PROTEINS AND ITS APPARENT REVERSAL*

II. HORSE SERUM PSEUDOGLOBULIN

BY HANS NEURATH, GERALD R. COOPER, AND JOHN 0. ERICKSON

(From the Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina)

(Received for publication, June 9, 1941)

In the preceding paper (2) the denaturation of horse serum al- bumin by urea and guanidine hydrochloride and its apparent re- versal were described. These studies have been extended in the present investigation to pseudoglobulin components with the object of determining any differences that may exist in the de- naturation process of the protein constituents of normal horse serum. Quantitative studies have been made possible by recent improvements in the experimental methods of isolating mono- disperse serum pseudoglobulin fractions (3, 4). Their known physical and chemical characteristics serve as a sensitive criterion for the extent to which denaturation can be reversed.

EXPERIMENTAL

Material

The method of preparation of monodisperse fractions of serum pseudoglobulins by means of fractional precipitation with am- monium sulfate under defined experimental conditions relative to protein concentration, pH, and salt concentration has been described in a previous publication (3). The present measure- ments were carried out by the technique already described (2) with the pseudoglobulins GI and GII, precipitable by ammonium sul- fate at pH 6.4 within the limits of 1.1 to 1.36 and 1.4 to 1.6 M

respectively. The molecular weights of GI and GII were found

* Presented at the Thirty-fifth annual meeting of the American Society of Biological Chemists at Chicago, April 15-19, 1941 (1).

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266 Denaturation of Proteins. II

by diffusion and viscosity measurements to be 170,000, in satis- factory agreement with the values of 165,000 obtained by Tiselius for electrophoretically isolated material (5), and of 178,000 found by Burk (6) from osmotic pressure measurements.’

Since it was found early in this work that no fundamental differences existed between the fractions GI and GII relative to the denaturation process as studied here, most of the measurements described below were carried out with the pseudoglobulin GII, as it was available in larger quantities.

Results

Denatured Pseudoglobulin in Presence of Urea and Guanidine Hydrochloride-The diffusion and viscosity of pseudoglobulin were measured in the presence of different amounts of urea and guani- dine hydrochloride in solutions containing 0.05 N acetate buffer and 0.2 M NaCl at pH 5.5. The results of the viscosity determina- tions carried out at protein concentrations between 0.1 and 1.2 per cent are plotted in Fig. 1.

As in the analogous studies on serum albumin (2), the relative viscosities increase with increasing concentration of the denaturing agent, the increase being greater for guanidine hydrochloride than for comparable concentrations of urea. The limiting slopes of the curves were determined from the intercept when qsP/c was plotted against c, where qsP is the specific viscosity and c the protein con- centration in weight per cent (7). Comparative measurements in the pressure viscometer showed the relative viscosities to be in- dependent of the velocity gradient within the region of 175 to 2000 sec.-‘.

Diffusion constants were measured in conjunction with the viscosity determinations. The results are summarized in Table I.

Analysis of the diffusion curves indicated the solutions to be essentially monodisperse in urea concentrations of 5 and 8 M, and in guanidine hydrochloride concentrations of 2 and 5.6 M. In 0.5 and 3 M guanidine hydrochloride there is some spreading of the values as determined by the method of successive analysis, in- dicating the presence of a small fraction of lower diffusion constant.

Calculations of apparent molecular shapes and molecular weights

1 Lower values, i.e. 142,000, have been reported by Cohn et al. (4) for material isolated by methods similar to those employed by the authors (3).

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Neurath, Cooper, and Erickson 267

from diffusion and viscosity data were carried out as described in the preceding paper (2) and are summarized in Table II’. There are also included values for the molecular shape, (b/~)~, calculated with the assumption of 33 per cent hydration. The empirical

l.36-.

1.28-

1.24-.

; z 0 1.20-- :: 5

I .os--

GUANIDINE HCL

0:2 0:a 016 6.6 1.0

PERCENT PROTEIN

JGo. 1. Relative viscosities of pseudoglobulin denatured by urea and guanidine hydrochloride, plotted against protein concentration in weight per cent. Open circles refer to 3 M guanidine hydrochloride, open squares to 8 M urea, open triangles to 5.6 M guanidine hydrochloride, solid circles to 0.5 M guanidine hydrochloride, solid squares to 5 M urea, and solid triangles to 2 M guanidine hydrochloride.

viscosity correction for the diffusion constant (2) was applied to the measurements in urea solutions and to the measurements in guanidine hydrochloride in concentrations higher than 2 M. In cases in which the solutions were found to be polydisperse, the limiting value of the diffusion constant was used for molecular weight calculations.

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TABLE I

Diffusion Constants of Denatured and “Reversibly”* and Irreversibly Denatured Pseudoglobulin

t = time in seconds; D = mean diffusion constant in sq. cm. per second; D’ = diffusion constant corrected for the viscosity of the solvent ((2) Equation 1); D,, D,, and Da are the diffusion constants calculated by the maximum height, standard deviation, and successive analysis methods, respectively. Unless otherwise indicated, each D3 value is the mean of about six values determined from evenly spaced parts of the diffusion curves (3).

,ry%Y--n~ t / D1 / Dz 1 D,

Pseudoglobulin in 0.5 M gusnidine HCl

Per cent sec.

10-V lo-’ 10-1

0.5 23,640 4.16 4.02 34,980 4.02 82,140 3.91 3.934.4:

Limiting value..... 4.4 X IO-’

D’.......... 4.6 X 1OP

Pseudoglobulin in 2 M guanidine HCl

0.55 24,720 2.76 32,640 2.86 41,580 2.87 73,380 2.79 2.89

Average. 2.85 X lo-’ f 0.05 D’. 3.19 X lo-’

- Pseudoglobulin in 3 M guanidine HCl

0.8 20,520 2.88 20,400 2.67 27,000 2.72 64,200 2.63 2.5833.01 81,120 2.75 2.61-3.0:

Limiting value.....

D’ 3.0 x 10-T 3.7 x 10-1

Pseudoglobulin in 5.0 M guanidine HCl

Average.. 2.50 X 10-r rfr 0.11 D’. 4.02 X lo-’

Pseudoglobulin in 5 M urea -_____--_

Per cent sec. 10-7 10-7 lo-’

1.2 38,640 2.97 75,000 2.75 2.72

104,400 2.77 164,100 2.65

Average. 2.76 X lo-’ f 0.12 D’. 3.73 X lo-’

Pseudoglobulin in 8 M ures,

1.2 86,400 1.76 109,440 1.68 1.79 132,420 1.75

51,165 1.65 79,380 1.73 1.89

103,680 1.62

Average. 1.70 x lo-’ zk 0.09 D’ 2.97 x 10-T

Pseudoglobulin, “reversibly” denatured by 8 M urea

0.7 35,760 4.56 4.50 45,600 4.44 59,940 4.49 71,940 4.61 4.55

Average. 4.53 x lo-’ f 0.12 D’. 4.69 X lo-’

Pseudoglobulin, irreversibly denatured by 7.5 iv urea, at pH 7.5

* See (2), foot-note 2.

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Neurath, Cooper, and Erickson 269

Distribution between L’Reversibly” and Irreversibly Denatured Pseudoglobulins-Removal of urea or guanidine hydrochloride by dialysis against distilled water in the cold resulted in partial pre- cipitation of the proteins. The precipitate was of gelatinous con- sistency varying in amount with the pH of the suspension and with

TABLE II

Molecular Constants oj” Native, .Denatu,red, and “Reversibly” Denatured Pseudoglobulins

qap/c = the limiting slope of the curves obtained when the specific viscosity is plotted against protein concentration; b/a = the ratio of the axes for a prolate ellipsoid, calculated with the Simha viscosity equation, solvation being neglected; (b/a), = the axial ratio calculated for 33 per cent hydration; D’ = the diffusion constant corrected for the viscosity of the solvent ((2) Equation 1); (J/fo) = l,he dissymmetry constant calculated from viscosity data; and M = the molecular weight calculated from diffusion and viscosity data.

Protein

Native..................... 6.60 In 0.5 M guanidine HCl.. 7.75 “ 2 M guanidine HCl 14.90 “ 3 “ “ I‘ 27.0 “ 5.6 M guanidine HCl 27.0 “ 5 M urea.. . . 14.0 ‘I 8 “ “ . . . . . . . . . . . . . . . 28.0

“Reversibly” denatured by 8 ivr urea ..___.._. ,__._ 5.90

7.2 5.2 8.1 6.0

13.0 10.0 19.0 15.0 19.0 15.0 12.4 9.3 19.5 15.4

10-7

4.75 4.6* 3.19 3.7* 4.02 3.73 2.97

6.6 4.7 4.69

1.39 170,000 1.44 170,000* 1.69 307,000 1.95 95,9OO*t 1.95 74,800j 1.66 152,ooot 1.98 170,000t

1.35 190,000

* The solution was somewhat polydisperse (see Table I). The values refer to the limiting diffusion constant determined by the method of suc- cessive analysis.

t Molecular weight calculated with the empirical correction for the diffusion constant (see the text).

temperature. Maximum yield was obtained when solutions were adjusted to pH 6.0 and stored at about 0”.

The quantitative distribution between insoluble (irreversibly denatured) and soluble (“reversibly” denatured) pseudoglobulin was studied as a function of the concentration of the denaturing agent originally present. 10 cc. samples of 2 per cent protein in 2, 4, 6, 7, and 8 M urea or guanidine hydrochloride were prepared

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270 Denaturation of Proteins. II

and, after 12 hours, dialyzed in the ice box against running distilled water until free from salt. The solutions were next adjusted to pH 6.0 and stored overnight at 0”. The precipitates were then centrifuged, washed once with distilled water, dissolved in a phosphate buffer of pH 7.1, and made to volume. The super- natants and washings were likewise made to volume and the pro-

9 I) 8~ t 5 0 lo- 3 m 2 60- w 2 $ so- z p 2 40-

/-

GUANIDINE HCL

I PSEUDOGLOBULIN

q//J// , , , 2 4 6 8 IO 12

MOLARITY OF UREA OR GUANIDINE HCL

FIG. 2. The fraction of total pseudoglobulin irreversibly denatured after dialysis, plotted against the molarity of urea or guanidine hydrochloride at which denaturation occurred. Open circles refer to urea, solid circles to guanidine hydrochloride,

tein concentrations in the respective fractions determined with the Koch-McMeekin method (8). The results of these measurements, carried out in triplicate, are given in Fig. 2 in which the fraction of total protein irreversibly denatured is plotted against the concen- tration of urea, or guanidine hydrochloride originally present.

“Reversibly” Denatured Pseudoglobulin--In the following ex- periments, a 2 per cent protein solution was denatured by 8 M urea

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Neurath, Cooper, and Erickson 271

and, after standing for 24 hours, urea was removed by dialysis. Diffusion measurements on the supernatant solution, obtained after precipitation of the irreversibly denatured protein at pH 6.0, indi- cated the material to be polydisperse. The diffusion constants, measured in the presence of an acetate buffer of pH 5.5, varied be- tween 3.8 X lop7 near the peak of the diffusion curves and 4.6 X lo-’ in the lower regions. The limiting slope of the viscosity curves, qsp/c, was found to be higher than that of the native globu- lin; i.e., 7.90 as compared with 6.60.

For further purification, the proteins contained in the super- natant solution were subjected to fractional precipitation with ammonium sulfate, according to the method employed for the purification of the native protein: the protein concentration was adjusted to 3 per cent, the pH to 6.4, and the ammonium sulfate concentration gradually increased to 1.1 M. At this point the solution became slightly opalescent and when more ammonium sulfate was added, up to 1.36 M, a precipitate settled out. After filtration, the salt concentration was raised to 1.6 M; the precipi- tate collected by centrifugation, dialyzed, and, after removal of traces of euglobulin by pH adjustment to 6.2 and 5.0, used for diffusion and viscosity measurements in the presence of a 0.05 M acetate buffer, pH 5.5, containing 0.2 M NaCl.

The results of the diffusion measurements are listed in Table I. The material proved to be monodisperse with a diffusion constant of D = 4.69 X 1O-7 with a standard deviation of the mean of SO.12 x 10-7. The limiting slope of the viscosity curves, il- lustrated in Fig. 3, was 5.90 as compared with 6.60 for native material. The molecular weight of 190,000 calculated from these data is somewhat higher than that found for the native material (Table I).

Irreversibly Denatured Pseudoglobulins-The fraction which pre- cipitated upon adjustment of the pH to 6.0, following removal of 8 M urea by dialysis, was considered to be irreversibly denatured material. For further purification, it was dissolved by acidifying to pH 4.0 and freed from any insoluble residue by filtration. In a concentration of 2 per cent protein, the solution was highly viscous and appeared to exhibit thixotropic properties. It did not show double refraction of flow in the apparatus described by Edsall and Mehl (9). The protein was reprecipitated by adjustment of the

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272 Denaturation of Proteins. II

pH to 6.0 and then redissolved at the desired pH. Addition of the buffer components by dialysis resulted in partial precipitation. The results of the viscosity measurements carried out in a 0.05 N acetate buffer at pH 4.12, containing 0.1 N NaCl, and in a 0.02 N veronal-acetate buffer at pH 7.5, containing 0.1 N NaCI, are shown in Fig. 3. The relative viscosities were also measured in t,he pressure viscometers and found to be independent of the veloc-

? -- 1.24 ,

!

DENATURED

> 1.20 c in 0

0 0.2 0.4 0.6 0.8 1.0 1.2

PERCENT PROTEIN

FIG. 3. Relative viscosities of irreversibly and “reversibly” denatured pseudoglobulin, plotted against the protein concentration in weight per cent. Triangles refer to irreversibly denatured protein at pH 4.1, squares to the same protein at pH 7.5, circles to “reversibly” denatured protein, purified by fractional precipitation with ammonium sulfate (see the text). For comparison, the slope of the viscosity curve of the native protein is also indicated.

ity gradient? between 400 and 2500 sec.-l. The rate of diffusion, like the viscosity, was a function of pH. At pH 4.12 no measurable rate could be observed after 72 hours diffusion of a 0.3 per cent solution, indicating gel formation. At pH 7.5, the calculated mean diffusion constant was considerably lower than that of the de-

2 There is little doubt, however, that structural viscosity may be de- tected at very low velocity gradients. Such measurements in a modified Couette apparatus are under way and will be reported elsewhere.

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Neurath, Cooper, and Erickson 273

natured protein in 8 M urea. The calculated D3 values varied widely from each other, probably due to restriction of free diffusion (Table I).

DISCUSSION

The denaturing effect of urea on pseudoglobulin is analogous to that observed for serum albumin. The apparent molecular asymmetry increases with increasing concentration of urea, while the molecular weight remains essentially unchanged. This latter finding is in agreement with the osmotic pressure measurements of Burk (6). The solutions of the denatured protein in urea are monodisperse, indicative of a uniform action of urea on all pseudo- globulin molecules.

The effects produced by guanidine hydrochloride are more com- plex. In 0.5 M concentration, guanidine produces only minor changes in apparent molecular shape and no changes in molecular weight. Diffusion measurements indicate, however, the presence of material of higher molecular weight, In 2 M solution, the ap- parent molecular asymmetry of the protein is markedly increased and the molecular weight is nearly twice that of the native protein.

When the guanidine hydrochloride concentration is increased to 3 M, the molecular asymmetry becomes drastically increased and about equal to that produced by 8 M urea solutions. The diffusion constant, however, does not decrease in proportion but, on the contrary, increases. The mean molecular weight, calculated from the limiting values of ~~~/c and D is about one-half of that of the native protein. Further increase in guanidine hydrochloride con- centration, to 5.6 M, produces no further changes in apparent molecular shape or molecular weight except that the solutions now become monodisperse (Table I). The action of guanidine hydro- chloride on pseudoglobulin is specific in that the molecule splits as it unfolds.3 There does not appear to exist any dimensional re- lation between the denatured whole molecules and the denatured halves such as has been observed with the splitting of native protein molecules (10). Simultaneous splitting and unfolding has also been observed in the denaturation of myogen by urea (11).

The observed relation between the concentration of denaturing

a See (2) foot-note 8.

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274 Denaturation of Proteins. II

agent and the fraction of total protein irreversibly denatured after dialysis is in qualitative accord with the analogous relation found for serum albumin, and may be interpreted on the basis of the statistical considerations discussed previously (2). Quantita- tively, however, these two sets of data differ from one another in that the limiting value of the fraction irreversibly denatured is 64 per cent for pseudoglobulin when denatured by urea and 84 per cent when denatured by guanidine hydrochloride, as compared with 15 per cent for serum albumin when denatured by either agent. This indicates fundamental differences in the intrinsic structure of these two proteins. The difference in maximum yield of irreversibly denatured pseudoglobulin produced by urea and guanidine hydrochloride is probably due to their different modes of action.

“Reversible” denaturation from 8 M urea solutions yields ma- terial of molecular size and shape similar to that of the native protein. Electrophoretic measurements on pseudoglobulin GI, “reversibly” denatured by 5 M urea, showed it to move with a single boundary on both the acid and alkaline side of the iso- electric point (12). The electrophoresis curves of the native and “reversibly” denatured material were practically indistinguishable from one another, except for differences in mobility.

The question of the true reversibility of denaturation demands a critical examination of the data at hand. It was shown that the protein remaining in solution after isoelectric precipitation of the irreversibly denatured fraction was polydisperse. However, the spread in diffusion constants was relatively narrow (3.8 to 4.6 X lo-‘), suggesting that polydispersity was not due to incomplete separation of “reversed” denatured and denatured protein, but rather to a gradation in molecular size or shape of the “reversed” denatured protein itself. This assumption finds support in Poison’s diffusion measurements on whole serum globulin (13) ob- tained by precipitation with half saturated ammonium sulfate, where the spread in calculated diffusion constants is comparable to that observed here for the unfractionated “reversed” denatured pseudoglobulin. Further support comes from the observed solu- bility properties. When “reversible” denaturation was carried out with pseudoglobulin GII, precipitation of the “reversibly” denatured protein was found to occur over the region of 1.1 and 1.6 M ammonium sulfate, whereas the salting-out region of the

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Neurath, Cooper, and Erickson 275

native material was confined to between 1.36 and 1.6 M. Pseudo- globulin GI, when subjected to ‘9eversible” clenaturation, started to precipitate at an ammonium sulfate concentration of 0.8 M, as compared with 1.1 M for the native protein, and continued up to 1.36 M. One may conclude, that “reversible” denaturation did not result in the restoration of a distinct molecular configuration, but rather in the formation of molecular entities of related intrinsic structures. This conclusion is also in accord with the ideas ex- pressed in a previous paper (12) concerning the continuous grada- tion in physical and chemical properties of the native globulins. A fraction approximating in properties the native material can be isolated from this mixture by subjecting it to fractional precipita- tion with salt under the same experimental conditions as have been used for the purification of the native protein. Further compara- tive studies of these fractions and of their immunological properties are under way.

The solubility properties of the irreversibly denatured protein are similar to those observed for the euglobulin fractions of normal horse serum as obtained by the method of isoelectric precipitation (14). Like these euglobulin components, the relative viscosity of this material is much higher than that of the native pseudo- globulin (15). Attempts to identify the protein in terms of molecular weight or shape were impeded by its anomalous be- havior in respect to diffusion.

The authors are indebted to the Rockefeller Foundation, to the Lederle Laboratories, Inc., and to the Duke University Research Council for support of this work.

SUMMARY

The clenaturation of horse serum pseudoglobulin by urea follows a similar pattern to that observed for serum albumin. The ap- parent molecular asymmetry, determined by viscosity memure- ments, increases with increasing concentrations of urea, whereas the diffusion constants decrease in proportion. The molecular weight remains unchanged during denaturation.

The denaturing effects produced by guanicline hydrochloride depend on the concentration. 2 M guanicline hydrochloride ap- pears to cause an aggregation of the protein molecules, the mean molecular weight being about twice that of the native protein. In

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276 Denaturation of Proteins. II

3 M solution the protein molecules split into halves as they unfold, whereas a further increase in the guanidine hydrochloride concen- tration, to 5.6 M, produces no additional changes in molecular size or shape except that the solutions become monodisperse.

Removal of the denaturing agent by dialysis causes a separation into two fractions which have been identified with “reversibly” and irreversibly denatured protein. The quantitative distribution between these two fractions is a function of the concentration of the denaturing agent. In equimolar concentrations, guanidine hy- drochloride leaves a larger fraction irreversibly denatured than does urea.

The “reversibly” denatured protein, purified by fractional pre- cipitation with ammonium sulfate, resembles, but is not identical with, the native protein in respect to molecular size, shape, and electrophoretic properties.

The irreversibly denatured protein resembles in respect to solu- bility and viscosity the euglobulin components as isolated by iso- electric precipitation from normal horse serum. The solutions ex- hibit a tendency for gel formation which is more pronounced on the acid side of the isoelectric point than on the alkaline side.

BIBLIOGRAPHY

1. Neurath, H., Cooper, G. R., and Erickson, J. O., Proc. Am. Sot. Biol. Chem., J. BioZ. Chem., 140, p. xcvi (1941).

2. Neurath, H., Cooper, G. R., and Erickson, J. O., J. Biol. Chem., 142, 249 (1942).

3. Neurath, H., Cooper, G. R., and Erickson, J. O., J. Biol. Chem., 138, 411 (1941).

4. Cohn, E. J., McMeekin, T. L., Oncley, J. L., Newell, J. M., and Hughes, W. L., J. Am. Chem. Sot., 62, 3386 (1940).

5. Tiselius, A., Biochem. J., 31, 1464 (1937). 6. Burk, N. F., J. Biol. Chem., 121, 373 (1937). 7. Mark, H., Physical chemistry of high polymeric systems, New York,

258 ff. (1940). 8. Koch, F. C., and McMeekin, T. L., J. Am. Chem. Sot., 46, 2066 (1924). 9. Edsall, J. T., and Mehl, J. W., J. Biol. Chem., 133, 409 (1940).

10. Neurath, H., J. Am. Chem. Sot., 61, 1841 (1939). 11. GralBn, N., Biochem. J., 33, 1342 (1939). 12. Sharp, D. G., Cooper, G. R., and Neurath, H., J. Biol. Chem., 142, 203

(1942). 13. Polson, A., KoZZoid-Z., 87, 149 (1939). 14. Green, A. A., J. Am. Chem. Sot., 60, 1108 (1938). 15. Fahey, K. R., and Green, A. A., J. Am. Chem. Sot., 60, 3039 (1938).

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EricksonHans Neurath, Gerald R. Cooper and John O.

HORSE SERUM PSEUDOGLOBULINAND ITS APPARENT REVERSAL: II.

THE DENATURATION OF PROTEINS

1942, 142:265-276.J. Biol. Chem. 

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