723
DENATURATION AND AGGREGATION OF OVALBUMIN
-
The Denaturation and Aggregation of Ovalbumin by Urea in Neutral Solutions
by Walter L. Gagen and John Holme The Procter and Gamble Company, M i a m i Valley Laboratories, Cincinnati $9, Ohio
(Received ApriE IS, 196s)
Physical properties, such as optical rotation, viscosity, sedimentation, ultraviolet absorption, and partial specific volume of ovalbumin exposed to urea in neutral solutions have been examined. In addition, the reactivity of SH groups in such solutions has been determined by amperometric titration. The changes which occur in these parameters during extended exposure to 6 M urea solutions have been shown to be related to the production of an aggregated form of the ovalbumin macromolecule. Although indirect evidence (e.g., increased levorotation) might suggest that the aggregated form has as its precursor a denatured species, no direct evidence for such a species in urea solutions was observed. The conformation of native ovalbumin in urea solutions is not markedly different from its conformation in simple salt solutions. The significant reductions in intrinsic sedimentation coefficients noted in such solutions are adequately described by changes in selective solvation parameters and are not indicative of changes in molecular asymmetry.
htroduetion The examination of changes in various physical and chemical properties that may be related to the denaturation and aggregation of ovalbumin in urea solutions has drawn tlie attention of numerous workers. The changes in optical rotation, viscosity, and the gelation of ovalbumin during exposure to various concentrations of urea have been studied by Simpson and Kauzmann,l Frensdorff, et a1.,2 and Steven and Tristram.a The latter workers, in addition, have measured changes in molar extinction coefficients a t 295 mb and, after iodination, at 310 mw. Imahori4and Schellman and S~hellrnan~have studied tke optical rotatory dispersion of ovalbumin in concentrated urea solutions, while partial specific volume measurements in such systems have been made by McKenzie, et a1.,6 and Charlwood.7 The volume changes accompanying the exposure of this protein to urea have been measured dilatometrically by Christensen8 and Simpson and Kauzmann.' Generally, these and other similar studies have been interpreted to indicate the existence of a denatured species of ovalbumin in concentrated urea solutioris, although Imahori4 has found that p-form association can and does occur in such solutions. Recent studies by Holme9 on the thermal denatura-
tion of ovalbumin have shown, however, that only native and aggregated forms of ovalbumin are found in heated solutions. The present study was initiated in an effort to determine whether such was also the case for urea denaturation, and if not, to isolate and characterize the denatured species.
Experimental Materials. A single lot of crystalline, lyophilized ovalbumin, purchased from Worthington Biochemical Corp., was used throughout. Reagent grade urea and buffer salts were used without further purification. Preparation of Ovalbumin- Urea Solutions. Ovalbumin was dissolved in distilled water (previously ~
~
~~
(1) R. B. Simpson and W Kauzmann, J . Am. Chem. Soc., 75, 5139 (1953). (2) H . K . Frensdorff, W. T. Watson, and W Kauzmann, ibid., 75, 5157 (1953). (3) F. 8. Steven and G. R. Tristram, Biochem, J., 73, 86 (1959). (4) K. Imahori, Biochim. Biophys. Acta, 37, 336 (1960). (5) 3. A. Schellman a n d C. Schellman, Compt. rend. traa. lab. Cadsbcrg, Ser. chim., 30, 363 (1958). (6) H. A. McKenzie, M . B. Smith, a n d R. G. Wake, Nature, 176, 738 (1955). (7) P. A. Charlwood, J . Am. Chem. SOC.,79, 776 (1957). (8) L. K. Christensen, Compt. rend. traa. lab. Carlsberg, Ser, chim., 28, 37 (1952). (9) J. Hoime, J . Fhys. Chem., 67, 782 (1983).
Volume 68, Number 4
April, 1964
724
WALTERL. GAGENAND JOHN HOLME
adjusted to pH 7.0) or phosphate buffer (pH 7.0, ionic strength 0.1). To this was added an equal volume of urea solution, pH 7.0, freshly prepared to avoid the complications of cyanate formation.’O The solutions were then mixed by very gentle stirring. The final concentrations of ovalbumin and urea are given in the individual experiments. Protein concentrations were determined by semimicro Kjeldahl nitrogen analysis (% N X 6.3) in the absence of urea, and by differential refractometry in the presence of urea. Density. Solution densities were measured a t 30 f 0.02’ using 25-ml. pycnometers. All samples were equilibrated in a constant temperature bath for about 30 min. prior to weighing. Partial Specific Volume. The conventional pycnometric technique” and the ultracentrifugal technique proposed by Martin, et aE.,lZ were both used for the determination of partial specific volumes. Using the conventional technique, the apparent partial specific volume, 4, at ovalbumin concentrations from 0.23 to 1.80% was calculated from the equationla
’ [I - -I-:
($=-
PS
where ps is the density of the solvent, A p is the increment in density due to the solute, and c is the solute concentration in g./ml. Since 4 was observed to be independent of ovalbumin concentration, the partial specific volume, P, was determined as the average of the 4values determined a t the various concentrations. Sedimentation measurements in HzO arid DzO (at an ovalbumin concentxation of 0.25%) permitted calculation of the partial specific volume by the equation of Martin, et aZ.l2
where 71, pl, and SIare the viscosity, density, and sedimentation coefficients, respectively, in HzO and qz, pz, and Sz are the comparable values in DZO. A value for k , the ratio of the molecular weights in the two media, 1.015, was calculated from the amino acid composition. Viscosity. Relative viscosities were determined from the product of the ratio of flow times for solution and solvent and the ratio of their densities, in the usual manner. Flow times were determined a t 30 f 0.2’ using an Ostwald-Fenske viscometer having a flow time for phosphate buffer of 59.4 sec. Viscosities are expressed, throughout this paper, in units of (g./ The Journal of Physical Chemistry
100 ml.)-I. Kinetic correction factors were found to be negligible and were therefore omitted. Optical Rotation. A Rudolph precision polarimeter and a equipped with a mercury light source (5461 water-jacketed 1-dm. cell maintained a t 30 * 0.2’ was used for all optical rotation measurements. Ultracentrifugation. Sedimentation analyses were made using a Spinco Model E ultracentrifuge equipped with temperature control and a phase-plate schlieren optical system. Sedimentation coefficients were determined in the usual manner14 from plots of In X€I us. time, where XH has been calculated from the maximal ordinate of the peak position. Peak areas were determined, by planimetry, from tracings of the schlieren patterns that had been enlarged fivefold. Relative concentrations mere calculated from the peak areas after correction for radial dilution14 and JohnstonO g s t o ~ i ’ *effects. ~~~ The correction of sedimentation coefficients to standard conditions is treated in a later section. Ultraviolet Absorption Spectra. The ultraviolet absorption spectra were obtained using a Beckman DU spectrophotometer with matched 1.OO-cm. silica cells. In addition, a limited number of spectra ‘were obtained using a Cary Model 14 recording spectrophotometer with matched 1.00-cm. cells. Sulfhydryl Determinations. Sulfhydryl groups were measured in TRIS buffer by amperometric titration^'^^'' utilizing silver- or mercury-plated rotating platinum electrodes operating a t potentials of -0.1 and -0.2 v./ v., respectively. Titrations were carried out with M silver nitrate or 10-3 M mercuric chloride solutions.
w.)
Results and Discussion Properties of Native Ovalbumin in Urea Solutions. Obviously, the definition of a denatured state of a protein macromolecule in solution requires previous definition of the macromolecule’s native state. In this study, the native state was defined as that conforma(10) G. R. Stark, W’. H. Stein, and S. Moore, J . Bid. Chem., 235, 3177 (1960).
(11) T. Svedberg and K. 0. Pedersen. “The Ultracentrifuge.” Oxford University Press, London, 1940. (12) W. G. Martin, W. H. Cook, and C. A. Winkler, Can. J . Chem.. 34, 809 (1956). (13) E. F. Casassa and H. Eisenberg, J . P h y s . Chem., 6 5 , 427 (1961). (14) H . K . Schachman, “Ultracentrifugation in Biochemistry,” Academic Press, New York, N. Y., 1959.
(15) J . P. Johnston and 4 . G. Ogston, Trans. Faraday SOC.,4 2 , 789 (1946).
(16) R. E. Benesch, H. A. Lardy, and R. Benesch, J . Biol. Chem., 216, 663 (1955) (17) I. M. Kolthoti, W. Strioks, and L. Morren, Anal. Chem., 26, 366 (1954).
725
DENATURATIOX AND AGGREGATIONOF OVALBUMIN
can be corrected for selective solvation through the substitution of an arbitrarily defined parameter, P h (designated in this paper as the hydrodynamic or effective partial specific volume), for P in the standard conditions correction equation. The parameter v h i s defined, in hydrodynamic terms, as the reciprocal of the medium density a t which the product ys = 0; i.e. l’h = l / p ”
F i g u r e 1. Concentration d e p e n d e n c e of sedimentation for o v a l b u m i n in 1, 3, 4,a n d 6 iM urea-water solutions. s20,w values corrected i n usual m a n n e r ( p = 0.750; see t e x t ) .
tion naturally assumed by the ovalbumin macromolecule immediately after dissolution in urea solution. I n order to define such a state in terms of physical parameters, the properties of ovalbumin immediately after dissolution (or as soon thereafter as experimentally possible) were studied as functions of both urea and protein concentration. a. Viscosity. The reduced viscosities of Ovalbumin in 1-6 M urea solutions were measured a t various protein concentrations from 0.23 to 1.80%. The intrinsic viscosity (i-e., the reduced viscosity extrapolated to infinite dilution) was found to be 0.040 + 0.005 regardless of urea concentration. b. Optical Rotation. From measurements of the optical rotation of ovalbumin (at 5461 I.)as a function of protein concentration in 4 and 6 M urea solutions, it was found that the specific rotation increased only slightly with protein concentration. The specific rotation a t infinite dilution was -35 f 3’. c. Sedimentation. The concentration dependence of sedimentation for ovalbumin in urea solutions was found to be influenced markedly by the urea concen tration. If one corrects the sedimentation coefficients to the usual standard condition^,^'^^^ the plots shown in Fig. 1 are obtained. Obviously, either the intrinsic sedimentation coefficiient is lowered in the presence of urea or the standard conditions correction is not adequate. I n attempting to choose between these alterna-. tives, one is immediately confronted with the problem of ‘(correcting” sedimentation coefficients to “standard conditions.” Schachman and Lauffer18 have shown, in studies on tobacco mosaic virus in 40% sucrose solutions, that selectiive solvation can lead to serious errors in the correction of sedimentation data. As pointed out by these workers, sedimentation data
where po is the medium density a t 0s = 0 (obtained from the linear plot of ys us. p for the protein in solvents of different viscosity and density). Calculation of V b was a,ccomplished by sedimentation of ovalbumin in HzO and D20 containing 3-6 M urea according to the technique of Martin, et al.l2 By this technique a value of 0.80 ==! 0.01 was found for l‘h. If this value for the eflective partial speciF-c volume is substituted for P (the thermodynamic partial specific volume) in the usual standard conditions correction equation, the sedimentation data shown in Fig. 1 extrapolate to approximately the sanae value (3.5 svedbergs) a t all urea concentrations. Additional studies concerning the parameter p h and the effect of various additives upon this parameter are now in progress but, for the present study, one need not be disconcerted by the concept of visualizing p h as an efective partial specific volume since an alternative procedure for treating the present data is available. The equation for sedimentation in a mixed-solvent system
Nfs
=
M,(1 - P,p)
+ aMi(1 - P i p )
permits the calculation of sedimentation coefficients in such systems if the selective interaction parameter, a , is known. As shown by Schachman,14 this parameter can be obtained in a manner similar to that described for V h determinations (z.e., from plots of 7s us. p ) . Thus, as an alternative to the concept of an effective partial specific volume, one can determine the selective interaction parameter a (in this instance, 1.39 X lo3moles/mole), use this value in the equation for sedimentation in a mixed-solvent system, and describe the characteristics of native ovalbumin in such systems solely in terms of selective interactions. This procedure leads to intercepts ( s ~ , , , ~similar ~) to those shown in Fig. 1. The ‘LP-functioii” of Scheraga and Mandelkeriz, l9 which combines data from intrinsic viscosity and sedi(18) H. K. Schachman and M. A. Lauffer. J . Am. Chem. Soc., 72, 4288 (1950). (19) H. A. Scheraga and L. Mandelkern, ibid., 75, 179 (1953)
Volume 68, Number 4
April, 1964
726
mentation measurements, allows one to describe more adequately the conformation of a macromolecule in solution (in terms of an effective ellipsoid of revolution) than does either measurement alone. Calculation of &functions for ovalbumin in 1-6 A4 urea solutions yields values, at all urea concentrations, not unlike those observed in simple salt solutions (Le., -2.4 X IO5). Although the relationship between the effective ellipsoid and the actual conformation of the macromolecule in solution cannot be ascertained from these measurements, it seems obvious that nothing resembling a major conformational change occurs upon the introduction of urea to a neutral solution of ovalbumin. Hence, the decreased intrinsic sedimentation coefficients noted in urea solutions evidently result from factors associated with Vt,and not from changes in molecular asymmetry. d. Partial Specific Volunze. The partial specific volume of ovalbumin in HzO was determined pycnometrically to be 0.752. In excellent agreement with this was the value of 0.750 obtained by the ultracentrifugal method. However, in 3 M urea, solution, a partial specific volume of 0.730 f 0.025 was found by the pycnometric method, while the ultracentrifugal method, as mentioned in the previous section, yielded a value of 0.80 f 0.01, presumably due to selective solvation effects. e. Sulfhydryl. The sulfhydryl content of ovalbumin in HzO and 4 and 8 M urea solutions was found to be 3.9, 4.4, and 4.4 moles of SH/mole of ovalbumin, respectively, when determined by mercurimetric amperometric titration. On the other hand, argentimetric titrations yielded a value of 3.9 moles of SH/mole of ovalbumin, regardless of urea concentration. f. Discussion of the Native State. An isotropically expanded or swollen state has been observed in the urea denaturation of bovine mercaptalbumin by Kay and Edsall,20 for bovine serum albumin by Doty and Katz,21 for bovine fibrinogen by Scheraga, et aLlZ2 and has been suggested by Scheraga and Mandelkern’ as an explanation for the diffusion and viscosity behavior of horse serum albumin noted by Neurath and S a ~ m .The ~ ~increased reactivity of the SH groups of ovalbumin to Hg+2 ions might suggest that similar swelling behavior also occurs in urea solutions. (The failure of Ag+ ions to react with such SH groups could be the result of steric hindrances imposed upon the somewhat larger Ag+ ions.) H a v e r , such expansion or swelling would be expected to manifest itself, assuming the shape factor remains constant, as an increase in the intrinsic viscosity. Within the limits of experimental error, no obvious differences between the inThe Journal of Phyaical Chemistry
WALTERL. GAGENAND JOHN HOLME
I
90.0
I
/
60””1/Y 50.0
30.0
0
-.300
;200
,000 Id00 2000 3000 4600 5600 6600 7000 6600 9000 l0,oOO TIME (minutes)
Figure 2. The effect of exposure time on the specific rotation and reduced viscosity of ovalbumin in 6 M urea-water.
trinsic viscosity of ovalbumin in simple salt solutions and that in urea solutions have been noted. From these various observations, it would appear that native ovalbumin in urea solution at neutral pH assumes a conformation not markedly different from its conformation in simple salt solutions. The significant reductions in the intrinsic sedimentation coefficients noted in such solutions are readily understood in terms of selective solvation effects and are not indicative of changes in molecular asymmetry. Properties of Ovalbumin during Extended Exposure to 4 M Urea Solution. The specific rotation and reduced viscosity of ovalbumin at a concentration of 1.80% in 4 M urea-water a t pH 7.0 were determined at various time intervals during an extended period of exposure a t 30 f 0,Zo. The specific rotation was -35 f 2’ and the reduced viscosity was 0.038 f 0.003 during the entire 0-348 min. observational period. Studies of a similar nature on ovalbumin in 4 M urea-phosphate buffer a t pH 7.0 yielded results not unlike those obtained with 4 M urea-water. Evidently, the presence of 4 M urea in neutral solutions of ovalbumin produces no marked time-dependent changes in the optical rotation and viscosity of such solutions. The present data, together with data reported by others,l-a indicate that the native state of (20) C. M. Kay and J. T. Edsall, Arch. Biochem. Biophys., 65, 354 (1956). (21)P. Doty and S. Katz, Abstracts, 118th National Meeting of the American Chemical Society, Chicago, Ill., September, 1950, p. 14C. (22) H.A. Scheraga, W. R. Carroll, L. F. Nims, E. Sutton. J. K. Backus, and J. M.Saunders J . Polymer Sci.. 14, 427 (1954). (23) H.Neurath and A. M.Saum, J . Biol. Chem., 128,347 (1939).
DENATURATION AND AGGREGATION OF OVALBUMIN
727
Table 1: Sedimentation Characteristics of 1.8% Ovalbumin during Extended Exposure to 6 M Urea-Watei Exvwum time. mi".
25 105a 285= 717 1,085 1,228 1,254 1,444 1,862 7,214 11,588
_-__-v
m.,,. s
0.75
wedbewe---
_ _ ___
-
Vh
= 0 -.
Component A
Component B
Comvoncnt A
Component B
2.09 2.16 1.91 2.26 2.43 2.00 2.07 -1.9 -1.9
...
...
... ...
2.92 2.58 3.05 3.28 2.70 2.80 -2.6 -2.6
... ...
. . .b
. . .*
Indications of heteroKeneity.
-2.8 3.29 2.92 2.76 2.61 2.60 2.21 2.03
. . .b . . .b
... -3.8 4.43 3.95 3.73 3.53 3.51 2.99 2.74
--
Relatire eoneentration. %-Component A Component B
100 -100 -100 56 45 22 18 14 10 v. sm. v. am.
... ... ...
44 55 78 82 86 90 -100
-100
* Values not determinable.
ovalbumin in urea solutions is stable for prolonged time intervals if the urea concentration is less than H M . Properties of Ovalbumin during Extended Exposure lo 6 M Urea Solution. a. Optical Rotation, Viscosity, and Sedimentation. Denaturation of proteins by reagents such as urea is generally considered to involve a rather marked unfolding of the naturally-occurring folded macromolecular structure leading to increases in levorotation and reduced viscosity. Aggregation or dissociation into subunits is frequently observed, but such processes are generally considered to be secondary phenomena. The specific rotation, reduced viscosity, and sedimentation characteristics of ovalbumin at a concentration of l.80To in 6 M urea-water and 6 M urea-phosphate buffer at pH 7.0 were examined at various time intervals during an extensive period of exposure a t 30 0.2'. As shown in Fig. 2, the changes in specific rotation and reduced viscosity in 6 M urea-water solutions take place in two fairly distinct stages: (1) a relatively rapid, initial stage, during which both the specific rotation and reduced viscosity increase at rather rapid rates, lasting for about 1500-2000 min.,
*
Figure 3. Sedimentation patterns for ovalbumin in 6 M ureawater at pH 7.0; speed 59,780 1.p.m.; bar angle = 60'; aamplea exposed to urea solution, prior to aedimentation analysis, for (A) 24min.; (B) 1085min.; (C) 1254min.; and(D) 11,588min.
and (2) a relatively slow, secondary stage during which the reduced viscosity continues to increase, although a t a somewhat slower rate, after the specific rotation values have reached a plateau. The data for ovalbumin in 6 M urea-phosphate buffer are similar in most respects to those obtained for 6 M urea-water solutions. The primary difference would seem to be that the increases in these parameters occur at a slightly diminished rate and reach lower final values in the presence of phosphate ions. The sedimentation characteristics of l.8Y0 ovalbumin in 6 M urea-water solutions show rather marked changes during exposure. Initially, a single, relatively symmetrical, slow-moving boundary, as shown in Fig. 3A, is observed. After progressively longer standing intervals, sedimentation patterns show increasing boundary asymmetry until, after approximately 700 min., a second, slightly faster-moving boundary is resolved (e.g., Fig. 3B). The relative concentration of this faster-moving component progressively increases while that of the slower-moving component decreases (see Fig. 3C), indicating conversion of the slower species to the faster species. Continued exposure yields sedimentation patterns, such as that shown in Fig. 3D, exhibiting an increased sharpening of the faster-moving boundary, and eventually a single, hyper-sharp boundary of gradually decreasing sedimentation rate. This hyper-sharpness and the decreasing sedimentation rate are typical of gels and gel-like materials and are not unexpected in such viscous solutions. The sedimentation data, corrected to standard conditions using Y = 0.75 and Yh = 0.80, are tabulated in Table I. The aggregation process begins immediately and closely parallels in extent the changes in optical rotation
WALTERL. GAGENAND JOHN HOLME
728
Table I1 : Sedimentation Characteristics of 1.8% Ovalbumin during Extended Exposure to 6 M Urea-Phosphate Exposure time, min.
26 1087 1406 2843 7230
________--______ v = 0.75-----Component A
.svedbergs--------
------...
Vi, = 0 . SO------
Component B
Component C
Component A
. . *
...
2.85 2.98 3.07
2.11 2.20 2.27
3.81 3.81 3.96 3.64
... ...
... >3.8 4.56 4.18
---------
-
... ...
and viscosity which occur as a function of exposure time, as shown in Fig. 4. Here sedimentation results are superimposed on the optical rotation and viscosity measurements as a function of exposure time. The results of sedimentation measurements in 6 IM urea-phosphate buffer, although less extensive in nature, apparently differ slightly from those observed in urea-water solutions. The resolution of two fastermoving species and slightly increased sedimentation coefficients, the latter presumably due to decreased solution viscosity and diminished charge effects, are the primary differences. These data are shown in Table 11. Several additional sedimentation studies, tabulated in Table 111, were conducted on 1.8% ovalbumin exposed to 6 M urea-water, then dialyzed against phosphate buffer prior to sedimentation analyses. As expected, the relative concentration of the monomeric species progressively decreases with time in favor of an aggregated species. In a second series, a similar concentration of ovalbumin was exposed to 6 &I urea-water or 6 M urea-
Component B
Component C
...
...
---Relative Component A
100 40 15
...
5.16 5.14 5.35 4.92
>5.1 6.16 5.65
... ...
concentration, %--Component Component B C
..
..
60 60 55-60 48
25 40-45 52
..
Table I11 : Sedimentation Characteristics of 1.8% Ovalbumin in Phosphate Buffer after Extended Exposure to 6 M Urea-Water
----v
svedbergs
-
Exposure time, min.
Component A
140 1 082 11)OOO
3.2 3.4 3.4
)
--
Relative ooncentration, % ComComponent ponent A B
SZO,~,
= 0.750---
7-
Component B
-10.5 -13.5 -11.9
84 40 17
16 60 83
phosphate buffer for approximately 1085 min., then subjected to sedimentation analyses under various conditions of solvent environment. The results, shown in Table IV, indicate further the integrity of the two sedimenting species, their relative concentrations not being appreciably altered by mild variations in the solvent medium.
Table IV : Sedimentation Characteristics of 1.8% Ovalbumin in Various Media after Extended Exposure to 6 M Urea Solution
OMPPNENT A 2.6s (IO%) B 3.5s (90%)
60
OMPeNENT A-3.3s (45%) B-4.4s (55%)
Sample A
Sample B
Exposed @ 30° t o 6 M urea-PO4 for 1087 min.
Exposed @ 30" to 6 M urea-Hi0 for 1082 min.
I
j.
Sedimentation in 6 M urea-PO4: component 1 2.48 S (40%) Component 2 4.00S (60%)
4
50.0
A-2.8s (
