Allowance for thermodynamic nonideality and Donnan effects in binding studies. Activity coefficients of charged ligands in the presence of albumin.

A combination of ultrafiltration with either equilibrium dialysis or frontal gel chromatography has been used to evaluate the effects of thermodynamic nonideality in mixtures of bovine serum albumin and charged ligands. Studies with methyl orange, chlorpromazine and chromate as ligand all demonstrated inadequacy of the Donnan effect for description of the difference between the concentrations of free ligand in a mixture and the protein-free phase with which it is in dialysis equilibrium. On the basis of a quantitative relationship derived for the situation in which Donnan and thermodynamic nonideality effects both operate, values of the second virial coefficient for albumin and ligand have been determined. For albumin and either methyl orange or chlorpromazine the magnitude of this second virial coefficient has been rationalized on the statistical-mechanical basis of excluded volume. For the albumin-chromate system, however, the thermodynamic nonideality was manifested as a negative deviation from Raoult's Law, in keeping with the classical behaviour of electrolyte ions. From the viewpoint of the characterization of ligand binding a unique feature of the ultrafiltration/gel chromatography and ultrafiltration/equilibrium dialysis methods is their ability to define not only the binding function but also the activity coefficient of ligand for a given acceptor-ligand mixture. Consequently, irrespective of whether the ligand is charged or uncharged, the intrinsic binding constant that is determined is the thermodynamic parameter instead of the apparent value that is obtained from methods based on assumed thermodynamic ideality.