The pH Dependence of the Affinity, Kinetics, and Cooperativity of Ligand Binding to Carp Hemoglobin, Cyprinus carpio

Abstract The pH dependence of the equilibrium and kinetics of ligand binding to carp hemolysate and the purified hemoglobin components of carp hemolysate with O2 and CO has been studied. The affinity of carp hemoglobin for O2 at alkaline pH is 160 times that at acid pH. A large but significantly different pH effect on the affinity for CO is observed. pH also has different effects on the kinetics of the reactions with the two ligands. The change in the rate of O2 dissociation over the pH range studied is 10 times that found for the rate of CO replacement by NO; and as previously reported, the change in the rate of combination of CO is twice that of O2. The value of n in the Hill equation is pH-dependent for both ligands, being maximal at neutral pH and decreasing as the pH is shifted from neutrality. This decrease in the value of n is shown to be due not to heterogeneity in the carp hemoglobin but to be the result of an apparent decrease in the cooperativity of the ligand-binding reaction. At pH 5.6 and below, the value of n is 1.0 for CO and 0.75 for O2 indicating no cooperativity. Below pH 5.6 and above pH 8.0, all equilibrium and kinetic constants are found to be unaffected by further pH changes. These results and the low, constant values of n are consistent with the hypothesis that at acid pH, carp hemoglobin, whether liganded or not, remains in a single low affinity conformation, perhaps that normally associated with the deoxygenated state. The hemoglobin appears to approach a similar situation at alkaline pH where the molecule has a high affinity similar to that of the ligand-saturated state.