Allowance for the effect of protein charge in the characterization of nonideal solute self-association by sedimentation equilibrium

This theoretical investigation explores the use of statistical-mechanical approaches to characterize the reversible tetramerization of a protein monomer with the size and charge characteristics of serum albumin under conditions where consideration of nearest-neighbor interactions suffices to describe effects of thermodynamic non-ideality. Such analysis of simulated sedimentation equilibrium distributions points to the adequacy of both the scaled particle theory and potential-of-mean-force methods for determining the self-association constant. Although the latter method usually entails the assignment of a magnitude to monomer net charge, this requirement can be obviated to some extent by repeating the analysis for a range of monomer charges and identifying the most appropriate value as that associated with a minimum in the sum-of-squares-of-residuals (SSR) of the best-fit descriptions of the sedimentation equilibrium distribution. Reasonable estimates of the association constant are usually obtained from corresponding analyses of the same sedimentation equilibrium distributions with activity coefficients obtained by scaled particle theory, an approach which also involves the identification of parameters on the basis of a minimum in SSR. However, the value of monomer charge determined must be regarded as a curve-fitting parameter rather than a true measure of monomer charge. Similar qualifications are shown to prevail in the scaled particle theory approach, which also involves the identification of parameters (the effective monomer volume and the polymer/monomer volume ratio) on the basis of a minimum in SSR. We therefore recommend discontinuation of the practice whereby quite precise distinction between modes of self-association has been attempted on the grounds of the physical credibility of the magnitudes of these additional curve-fitting parameters.