Characterizing Reversible Protein Association at Moderately High Concentration Via Composition-Gradient Static Light Scattering

Analysis of weakly self-associating macromolecules at concentrations beyond a few g/L is challenging on account of the confounding effect of thermodynamic nonideality on the association signal. When the reversible association comprises only 1 or 2 oligomeric species in equilibrium with the monomer, the nonideality may be accounted for in a relatively rigorous manner, but if more association states are involved, the analysis becomes quite complex. We show that under reasonable assumptions, the nonideality in a composition-gradient static light scattering measurement may be accounted for in a simple fashion. The correction is applied to determining the stoichiometry and binding affinity of a protein previously characterized via sedimentation equilibrium and dynamic light scattering. The results of the new analysis are remarkably self-consistent and in line with the expectations for the form of self-association predicted previously from analysis of the surface residuals, establishing composition-gradient multi-angle static light scattering with nonideality corrections as a critical technology for characterizing associative interactions in concentrated solutions.