Iwuoha, E. I. and Okonjo, K. O.
(1993)
Role of the Relaxation of the Iron(ll1) Ion Spin States Equilibrium in the Kinetics of Ligand Binding to Methaemoglobin.
J. CHEM. SOC. FARADAY TRANS, 89 (21).
pp. 3921-3924.
Abstract
Temperature-jump experiments of the reaction of the thiocyanate ion with human aquomethaemoglobin have
been performed in the presence of a 10-fold excess of inositol hexakisphosphate (inositolP,). Two kinetic
phases corresponding to the a and /3 subunits were observed. Kinetic parameters of the reaction were evaluated
from the reciprocal relaxation times on the basis of a fast relaxation of the iron(iii) ion spin states equilibrium
before binding of the ligand. The association, ki,, and dissociation, k-i,, rate constants determined were:
k,, = 225 dm3 mol-' s-', k-aL = 1.52 s-', k,, = 2430 dm3 mol-' s-', k-pL = 6.51 s-' at 27"C, pH 6.44. There
was good agreement between the equilibrium constant of the ligand binding step determined by static methods
(Kequ = 204 & 11 dm3 mol-') and that evaluated from kinetic data [(KaLKpL)1'2 = 235 & 12 dm3 mol-'1. The
value ksJkaL = 11 obtained ensured proper separation of the two kinetic phases. Analyses of the subunit relaxation
amplitudes, a€,, , showed that inositolP, perturbed the absorption spectrum of the /3 subunits. This suggests
that in the presence of the organic phosphate, methaemoglobin behaves as a protein with independent
binding sites rather than as an allosteric molecule. The kinetic and relaxation amplitude spectral characteristics
of the subunits, in the presence of inositolP, have demonstrated that the kinetic dynamics are effectively decoupled in a stable tetramer.
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