Effect of sucrose on the dimerization of alpha-chymotrypsin. Allowance for thermodynamic nonideality arising from the presence of a small inert solute.

The space-filling effects of sucrose on the dimerization of alpha-chymotrypsin have been investigated by sedimentation equilibrium studies on the enzyme in acetate-chloride buffer, pH 3.9, I 0.2. From the extent of enhancement of the apparent dimerization constant in the presence of 0.05-0.16 M sucrose, it is concluded that this effect of thermodynamic nonideality finds quantitative explanation in terms of excluded volume. However, the suggested approximation that the radius of an inert small solute would be sufficiently small to be neglected in the calculation of covolumes (D.J. Winzor and P.R. Wills, Biophys. Chem. 25 (1986) 243) has not withstood the more stringent test afforded by the present study of alpha-chymotrypsin dimerization. A value of 0.34 nm for the effective thermodynamic radius of sucrose was inferred from the covolume for self-interaction obtained by frontal gel chromatography on Sephadex G-10 under the conditions of the ultracentrifugal studies. Finally, results of sedimentation equilibrium experiments on alpha-chymotrypsin in the presence of 0.1 M glycerol were also shown to be consistent with interpretation in terms of the model of space-filling effects entailing complete exclusion of small solute from the hydrated protein domain.