Temperature-jump method for determining the equilibrium constant of the iron spin states in human methaemoglobin A

Abstract

Spin equilibrium constants, K_spin, have been evaluated from chemical relaxtion amplitudes of temperature-jump experiments and equilibrium binding data for the reaction of thiocyanate ion with unmodified, active sulfydryl group (Cys F9 [93]β)-modified and inositol hexakisphosphate (inositol-P₆)-saturated methaemoglobins. The analyses were based on a scheme where a high-to-low spin transition of the iron(III) ion in methaemoglobin precedes the ligand-binding step. The results obtained show that chemical modification with cystamine increases the binding affinity of methaemoglobin, and perturbs the spin equilibrium (the latter is unaffected by iodoacetamide modification which reduces methaemoglobin binding affinity). This behaviour of modified methaemoglobins has been explained on the basis of Tanford–Kirkwood solvent accessibility theory. Inositol-P₆ diminished the binding affinity, but increased the K_spin of methaemoglobin as expected, which therefore, validates the temperature-jump method for K_spin determination.