Presence of a preferred anion-exchange binding site on cytochrome b5: structural and thermodynamic considerations.

A preferred chromatographic contact region for adsorption of recombinant soluble tryptic fragment of rat cytochrome b5 on the hydrophilic anion-exchanger Mono Q has been identified using conservative carboxylate-to-amide mutations of charged residues. Equilibrium adsorption isotherms were measured under conditions of full reversibility by high ionic strength, as confirmed by explicit mass balances performed for each experiment. Although cytochrome b5 displays several clusters of negative charge, mutations in one cluster consistently reduce binding affinity and the stoichiometric displacement parameter Z by much greater factors than do mutations in other areas of the molecule. Adsorption heterogeneity derived by fitting isotherms to the Hill equation is reduced by factors which reduce the overall affinity of adsorption. Van't Hoff analyses gave uniformly positive enthalpies of adsorption, and mutational changes in adsorption enthalpy were relatively independent of the site of mutation. These results suggest that enthalpy does not play a dominant role in either affinity or selectivity of anion-exchange adsorption in this system.