Determination of protein-ligand equilibriums by difference spectroscopy. Hemerythrin-ligand thermodynamic studies

Abstract

A difference spectrophotometric method for the rapid determination of equilibrium constants for protein-ligand interactions has been developed. The method requires no knowledge of the extinction coefficient of either reactants or products. The method allows rapid determination of the temperature dependence of a reaction and leads to rapid determination of thermodynamic parameters. The method was tested by following the interactions of ligands with hemerythrin, the nonheme iron, oxygen storage protein isolated from Phascolopsis gouldii. The reactions were studied at various temperatures and ionic strengths, and standard thermodynamic parameters were determined. The standard thermodynamic parameters for the conversion of metaquohemerythrin to methydroxyhemerythrin were .DELTA.H.degree. [enthalpy] = 5.8 .+-. 1.3 kcal mol-1 and .DELTA.S.degree. [entropy] = 11.5 .+-. 1.5 cal mol-1 deg-1. For the reacton of metaquohemerythrin with thiocyanate ion to produce metthiocyanatohemerythrin .DELTA.H.degree. = 13.0 .+-. 1.6 kcal mol-1 and .DELTA.S.degree. = -25.3 .+-. 5.5 cal mol-1 deg-1. For the reaction of thiocyanate ion with methydroxyhemerythrin .DELTA.H.degree. = 6.6 .+-. 0.8 kcal mol-1 and .DELTA.S.degree. = -38.3 .+-. 4.0 cal mol-1 deg-1. Perchlorate ion decreases the affinity of metaquohemerhythrin for thiocyanate ion. This is reflected in the entropy and enthalpy being more unfavorable for the reaction in the presence of perchlorate ion.