Allowance for thermodynamic non-ideality in the characterization of protein self-association by frontal exclusion chromatography: hemoglobin revisited.

This investigation re-examines theoretical aspects of the allowance for effects of thermodynamic non-ideality on the characterization of protein self-association by frontal exclusion chromatography, and thereby provides methods of analysis with greater thermodynamic rigor than those used previously. Their application is illustrated by reappraisal of published exclusion chromatography data for hemoglobin on the controlled-pore-glass matrix CPG-120. The equilibrium constant of 100/M that is obtained for dimerization of the alpha(2)beta(2) species by this means is also deduced from re-examination of published studies of concentrated hemoglobin solutions by osmotic pressure and sedimentation equilibrium methods.