A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.