ROLE OF THE RELAXATION OF THE IRON(III) ION SPIN STATES EQUILIBRIUM IN THE KINETICS OF LIGAND-BINDING TO METHEMOGLOBIN

Temperature-jump experiments of the reaction of the thiocyanate ion wi th human aquomethaemoglobin have been performed in the presence of a 1 0-fold excess of inositol hexakisphosphate (inositol-P6). Two kinetic phases corresponding to the alpha and beta subunits were observed. Kin etic parameters of the reaction were evaluated from the reciprocal rel axation times on the basis of a fast relaxation of the iron(III) ion s pin states equilibrium before binding of the ligand. The association, k(iL), and dissociation, k(-iL), rate constants determined were: k(alp haL) = 225 dm3 mol-1 s-1, k(-alphaL) = 1.52 s-1, k(betaL) = 2430 dm3 m ol-1 s-1, k(-betaL) = 6.51 s-1 at 27-degrees-C, pH 6.44. There was goo d agreement between the equilibrium constant of the ligand binding ste p determined by static methods (K(equ) = 204 +/- 11 dm3 mol-1) and tha t evaluated from kinetic data [(K(alphaL)K(betaL))1/2 = 235 +/- 12 dm3 mol-1]. The value k(betaL)/k(alphaL) = 11 obtained ensured proper sep aration of the two kinetic phases. Analyses of the subunit relaxation amplitudes, deltaE(iL), showed that inositol-P6 perturbed the absorpti on spectrum of the beta subunits. This suggests that in the presence o f the organic phosphate, methaemoglobin behaves as a protein with inde pendent binding sites rather than as an allosteric molecule. The kinet ic and relaxation amplitude spectral characteristics of the subunits, in the presence of inositol-P6 have demonstrated that the kinetic dyna mics are effectively decoupled in a stable tetramer.