Self-association of sperm whale metmyoglobin

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 234, No. 1, October, pp. 125-128, 1934

Self-Association

of Sperm Whale Metmyoglobin

LARRY D. WARD’ Department

of Biochemistry,

University

AND

DONALD J. WINZOR’

of Queensland, St. Lucia, Queens.!and 4067, Australia

Received March 1, 1934

The solution behavior of sperm whale metmyoglobin in 0.15 I phosphate-chloride buffer, pH 7.2, has been examined by sedimentation equilibrium, frontal gel chromatography, and sedimentation velocity. Results obtained from all three studies are shown to be consistent with a self-association model in which dimerization of the myoglobin is governed by an association equilibrium constant of 0.068 liter/g (580 M-‘)

at 20°C.

0 1994 Academic

Press, Inc.

The tendency of myoglobin to form dimers has been inferred from experimental studies in which solutions of the protein were supplemented with high concentrations of hemoglobin (1) or other inert proteins (2). Since those results are indeed most readily explained in terms of excluded volume effects on the thermodynamic activities of species coexisting in self-association equilibrium (3,4), the aim of the present investigation has been to determine whether reversible self-association of myoglobin is detectable by conventional physicochemical methods in the absence of any enhancement due to thermodynamic nonideality. To this end, the solution behavior of sperm whale metmyoglobin has been examined by frontal gel chromatography, as well as by the velocity and equilibrium techniques of ultracentrifugation. MATERIALS Crystalline was dissolved pH 7.2,10.155 0.140~ NaCl), against more

AND

METHODS

sperm whale metmyoglobin (Sigma) directly in phosphate-chloride buffer, (0.0017 M NaH2POI, 0.0043M NaaHPOI, and then dialyzed at 4’C for 16 h of the same buffer. Concentrations of

i Present address: Graduate Department of Biochemistry, Brandeis University, Waltham, Mass. 02254. 2To whom requests for reprints should be sent.

the dialyzed myoglobin solutions were then estimated spectrophotometrically on the basis of an absorption coefficient (A:‘:,) of 90 at 410 nm (5). Ultracentrifugation Sedimentation experiments were carried out at 20°C in a Spinco Model E ultracentrifuge fitted with electronic speed control. Speeds in the range 18,000-48,000 rpm were used in sedimentation equilibrium experiments, and velocity runs were performed at 60,000 rpm in a valve-type synthetic boundary cell. The Schlieren optical system was used for velocity sedimentation, whereas the solute distributions in equilibrium experiments were recorded as Rayleigh interferograms. These refractometric records in terms of Rayleigh fringes, J(r), were converted to weight concentrations (mg/ml) E(r), by dividing by 4.07, a factor obtained from synthetic boundary experiments with myoglobin solutions for which the concentration had been predetermined spectrophotometrically. Sedimentation equilibrium distributions were then subjected to Q(r) analysis (6) in order to determine the corresponding distribution of monomeric myoglobin, and the results analyzed in terms of the logarithmic form of the law of mass action for a two-state selfassociating system (nA P C). In sedimentation velocity experiments the boundaries obtained with myoglobin were sufficiently symmetrical for the sedimentation coefficient determined from the rate of migration of the Schlieren peak to be taken as S, the weight-average sedimentation coefficient. Allowance was made for radial dilution in determination of the corresponding myoglobin concentration, 6, from the loading concentration. The buffer density was measured with an Anton Paar DMA 60/602 precision density meter, and the relative viscosity with an Ostwald viscometer having a flow-time of 4 min for water; the partial specific volume of myoglobin was taken as 0.741 ml/g (7). 125

0003-9861/34 $3.00 Copyright All righta

0 1954 by Academic Press. Inc. of reproduction in any form reserved.

126

WARD

AND

WINZOR

Gel chromatography. Solutions of sperm whale metmyoglobin (0.2-4.8 mg/ml) were subjected to frontal gel chromatography (8) on a column of Sephadex G-75 (1.6 X 18.2 cm) equilibrated at 20°C with the 0.155 I phosphate-chloride buffer, pH 7.2. The column effluent, maintained at a flow rate of 22 ml/h, was collected in l-ml fractions, the precise volumes of which were determined by weight. Each fraction was then diluted appropriately (by weight) for spectrophotometric analysis at 410 nm, and the weight-average elution volume, v, was determined as the centroid (9) of the advancing elution profile. 0.00 0

RESULTS

AND

DISCUSSION

In this investigation the techniques of gel chromatography, sedimentation velocity, and sedimentation equilibrium have all provided evidence for the self-association of sperm whale metmyoglobin in 0.155 I phosphate-chloride buffer, pH 7.2. Since the last-named method yields the most unequivocal quantitative description of the phenomenon, the sedimentation equilibrium study is presented first, and the self-association model of myoglobin deduced therefrom is then used to interpret the results obtained by the two mass migration procedures.

1 F(rl

2

hg/ml)

3

-1.0 -0.1

0

0.4

0.1

LOO a,(‘)

FIG. 1. Analysis of sedimentation equilibrium experiments on sperm whale metmyoglobin in 0.155 I phosphate-chloride buffer, pH 7.2. (a) n(r) analysis (6) showing the extrapolation involved in determining the activity of monomer, U,(Q), corresponding to a selected total concentration E(Q), of 1.34 mg/ml. (b) Test of the results for conformity with a selfassociating system comprising monomer in equilibrium with a single higher polymer. Symbols denote experiments in which the angular velocities and loading concentrations were n , 18,000 rpm and 1.53 mg/mI; 0, 18,000 rpm and 2.44 mg/mI; and 0,20,000 rpm and 1.71 mg/ml.

that of 17,000 used by Minton and coworkers (1, 2), and with that of 17,800 Sedimentation Equilibrium Studies inferred from the sequence of myoglobin (12). (iii) The important point to note The results of sedimentation equilibrium experiments on sperm whale met- from Fig. la is the fact that the ordinate intercept, which defines the ratio al (rr)/ myoglobin in 0.155 I phosphate-chloride, pH 7.2, are summarized in Fig. 1, about E(rr), is significantly below unity; hence, which the following points are noted. (i) monomeric metmyoglobin is not the sole Figure la presents the Q(r) analysis (6) macromolecular species present. (iv) Figof results from three low-speed (10) ex- ure lb is a test of the results for conforperiments, and shows the extrapolation mity with an equilibrium system comrequired for evaluating the thermodyprising monomer and a single higher namic activity of monomer, ai( asso- polymer (nA P C). In this regard the ciated with the selected reference total coupling of concentrations and activities concentration, E(rr), of 1.34 mg/ml. (ii) In in the ordinate variable implies thermothat regard, evaluation of Q(r) is based dynamically ideal behavior, a reasonable on a magnitude of 4379 for the product approximation for relatively small globMi (1 - Vp), where Mi denotes the molec- ular proteins over the limited range (0.7ular weight of monomeric myoglobin and 2.9 mg/ml) of E(r) studied (13-16). (v) The p the solution density, this being the value results are clearly described adequately of the product obtained from the limiting by the line drawn with a slope of 2 (Fig. slope (E(r) < 0.05 mg/ml) of a plot of In lb), and thus conform with the logarithc(r) versus r2 from a high-speed (11) sed- mic form of the law of mass action for a imentation equilibrium experiment at reversibly dimerizing system. We there48,000 rpm. The consequent estimate of fore conclude that sperm whale metmyo17,100 for Ml is in good agreement with globin can be considered in terms of a

SELF-ASSOCIATION

OF SPERM

monomer-dimer system for which the association equilibrium constant, obtained from the ordinate intercept of Fig. lb, is 0.068 liter/g under the present conditions (pH 7.2,IO.l55,2O”C): a molar association constant of 580 M-~ is thereby indicated. Gel Chromatography of Mgoglobin The above detection of the reversible self-association of myoglobin in a relatively low concentration range(c < 3 mg/ ml) implies that the phenomenon should also be readily apparent from the mass migration methods (such as gel chromatography and sedimentation velocity) that are usually employed as a prelude to sedimentation equilibrium studies. This evidence of self-association is, indeed, readily obtained from frontal gel chromatography experiments on sperm whale metmyoglobin in 0.155 I phosphate-chloride, pH 7.2, there being a systematic inverse relationship between elution volume and solute

“‘5T. 2o.oL 0

J--d 0

1

1

2

I

I

2

3

t

(mu/ml)

4 E

h,,~l,

4

5

a

10

FIG. 2. Concentration dependence of (a) the elution volume on Sephadex G-75 and (b) the sedimentation coefficient of sperm whale metmyoglobin in 0.155 I phosphate-chloride, pH 7.2; the solid line denoting in each case the theoretical dependence predicted for a monomer-dimer system governed by an association constant of 0.068 liter/g (Fig. 1). 0, Results of the present study; 0, results taken from Fig. 2 of Breslow (20); Cl, result reported by Schachman and Edelstein (5).

WHALE

MYOGLOBIN

127

concentration (Fig. 2a). Extrapolation of the results to zero concentration signifies an elution volume of 21.0 ml for monomeric myoglobin, which corresponds to a partition coefficient, K,,, of 0.36, the value calculated by Laurent and Killander (17) from the results of Andrews (18) for myoglobin on Sephadex G-75. Moreover, by means of the usual (18) semilogarithmic calibration plot in terms of molecular weight, a value of 16.6 ml may be inferred for the elution volume of dimeric myoglobin. From the curve drawn in Fig. 2a, which is based on these two elution volumes and the association constant of 0.068 liter/g deduced from Fig. 1, it is evident that the gel chromatographic results provide quantitative substantiation of the self-association model of myoglobin inferred from the sedimentation equilibrium studies. Sedimentation Velocity Studies of Myoglobin The sedimentation velocity behavior of myoglobin has been the subject of several investigations, e.g., Refs. (19, 20); it is therefore surprising that the self-association has remained undetected for so long. Typically, myoglobin has been considered as a single macromolecular entity with a sedimentation coefficient (s&,,) of 2.0-2.2 S (19,20) from studies spanning a concentration range of 2 to 10 mg/ml. However, from investigations of more-dilute solutions (less than 0.1 mg/ml), Schachman and Edelstein (5) have obtained sedimentation coefficients (So,+,,)of 1.8 S; accordingly, further studies of the s-c dependence for myoglobin have been undertaken. Figure 2b presents the results of those experiments (O), together with the findings (Cl) of Schachman and Edelstein (5) and a typical set of published results (0), taken from Fig. 2 of Ref. (20). The curve is the theoretical relationship for a monomer-dimer system with an association constant of 0.068 liter/g (Fig. lb), values of 1.80 S and 2.75 S for the respective sedimentation coefficients (s!) of monomeric and dimeric species, and the relationship S = (2 s,c;}/E, where si = &l - gC). A value of 0.008 liter/g was assumed

128

WARD

AND

for g, the coefficient of concentration dependence (21), and the sedimentation coefficient of dimer was calculated from the expression si = s?(22/3)/df/f0)ZVthe magnitude of the frictional ratio for dimer, (f/fo)Z, being taken as that (1.04) of a prolate ellipsoid of revolution with an axial ratio of 2. Several points emerge from Fig. 2b. First, because of the relatively small magnitude of Sfor myoglobin, its measurement is prone to considerable experimental error, as indicated by the scatter of the present results. Second, there is no obvious disparity between the present results (0) and those (0) of Breslow (20), which presumably are subject to similar extents of uncertainty. Third, although the experimental results exhibit too much scatter to allow their use for defining the s-c dependencej they do not refute the concept of their description in terms of that for the monomer-dimer system deduced from the sedimentation equilibrium studies (Fig. 1). Finally, it is clear that the failure to detect the dimerization from earlier sedimentation velocity studies (19,20) reflected (a) the difficulties associated with accurate measurement of small sedimentation coefficients, and (b) the lack of measurements at concentrations below 2 mg/ml. In summary, the present investigation has established the existence of myoglobin as a monomer-dimer system, a conclusion that had previously been reached (1, 2) through considerations of thermodynamic nonideality in concentrated protein solutions. This more direct approach to the problem has not only confirmed the correctness of the qualitative interpretation (1,2) of earlier results in terms of excluded volume effects on a dimerizing system, but has also provided a quantitative description of the self-association phenomenon.

WINZOR ACKNOWLEDGMENTS The technical assistance of C. J. Leeder is gratefully acknowledged, as is the partial support of this investigation by the Australian Research Grants Scheme. L.D.W. was the recipient of a Commonwealth Postgraduate Award during the course of this investigation. REFERENCES 1. MINTON, A. P., AND LEWIS, M. S. (1981) Biqphys. Chem, 14,317-324. 2. WILF, J., AND MINTON, A. P. (1981) B&him Biophgs. Acta 670, 316-322. 3. NICHOL, L. W., OGSTON, A. G., AND WILLS, P. R. (1981) FEB.? L&t 126, 18-20. 4. MINTON, A. P. (1981) Biqdgmws 20,2093-2120. 5. SCHACHMAN, H. K., AND EDELSTEIN, S. J. (1966) Biochemistry 5.2681-2705. 6. MILTHORPE, B. K., JEFFREY, P. D., AND NICHOL, L. W. (1975) Biuphys. Chem 3.169-175. 7. THEORELL, H. (1934) B&hem 2. 268,46-54. 8. WINZOR, D. J., AND SCHERAGA, H. A. (1963) Biochemistry 2, 1263-1267. 9. LONGSWORTH, L. G. (1943) .I. Amer. Chem Sot 65,1755-1765. 10. VAN HOLDE, K. E., AND BALDWIN, R. L. (1958) J. Phys. Chem. 62, 734-743. 11. YPHANTIS, D. A. (1964) Biochemistry 3,297-317. 12. EDMLJNDSON, A. B. (1965) Nature (London) 205, 883-887. 13. OGSTON, A. G., AND WINZOR, D. J. (1975) .I Phys. Chem 79,2496-2500. 14. TELLAM, R., WINZOR, D. J., AND NICHOL, L. W. (1978) B&hem J. 173, 185-190. 15. TELLAM, R., DE JERSEY, J., AND WINZOR, D. J. (1979) Biochemistry 24, 5316-5321. 16. FORD, C. L., AND WINZOR, D. J. (1982) B&him. Biophys. Acta 756, 49-55. 17. LAURENT, T. C., AND KILLANDER, J. (1964) J. Chrowuztogr. 14.317-330. 18. ANDREWS, P. (1964) Biochem J. 91,221-233. 19. BANASZAK, L. J., EYLAR, E. H., AtiD GURD, F. R. N. (1963) J. Bid Chem 233, 1989-1994. 20. BRESLOW, E. (1964) J. Bid Chem. 239,486-496. 21. GILBERT, L. M., AND GILBERT, G. A. (1973) in Methods in Enzymology (Hirs, C. H. W., and Timsheff, S. N., eds.), Vol. 27, pp. 273-296, Academic Press, New York.