Studies of solute self-association by sedimentation equilibrium: allowancefor effects of thermodynamic non-ideality beyond the consequences of nearest-neighbor interactions

A sedimentation equilibrium study of a-chymotrypsin self-association in ace tate-chloride buffer, pH 4.1 I 0.05, has been used to illustrate determinat ion of a dimerization constant under conditions where thermodynamic non-ide ality is manifested beyond the consequences of nearest-neighbor interaction s. Because the expressions for the experimentally determinable interaction parameters comprise a mixture of equilibrium constant and excluded volume t erms, the assignment of reasonable magnitudes to the relevant virial coeffi cients describing non-associative cluster formation is essential for the ev aluation of a reliable estimate of the dimerization constant. Determination of these excluded volume parameters by numerical integration over the pote ntial-of-mean-force is shown to be preferable to their calculation by appro ximate analytical solutions of the integral for this relatively small enzym e monomer with high net charge (+ 10) under conditions of low ionic strengt h (0.05 M). (C) 2001 Elsevier Science B.V. All rights reserved.