Ei. Iwuoha et Ko. Okonjo, ROLE OF THE RELAXATION OF THE IRON(III) ION SPIN STATES EQUILIBRIUM IN THE KINETICS OF LIGAND-BINDING TO METHEMOGLOBIN, Journal of the Chemical Society. Faraday transactions, 89(21), 1993, pp. 3921-3924
Citations number
21
Categorie Soggetti
Chemistry Physical","Physics, Atomic, Molecular & Chemical
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.