The Effect of Inositol Hexaphosphate on the Kinetics of CO and O2 Binding by Human Hemoglobin

Abstract

Abstract The binding of inositol hexaphosphate (IHP) to oxyhemoglobin (oxy-Hb) was investigated by gel filtration and stopped flow kinetic methods. The stoichiometry of the complex is 1 IHP per hemoglobin tetramer in 0.05 m 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol (pH 7.0) containing 0.11 m NaCl and appears to approach 2 IHP per tetramer when the ionic strength is reduced to 0.01. The dissociation constant for the IHP-oxy-Hb complex is of the order of 10-6 m. Kinetic experiments conducted over a wide range of concentrations of oxy-Hb suggest, in addition, that the liganded hemoglobin dimer does not bind IHP. The complex of IHP with deoxyhemoglobin also exhibits a stoichiometry of 1 organic phosphate molecule per hemoglobin tetramer; the dissociation constant was not measured but must be less than 10-8 m based on the large inhibitory effect of IHP on oxygen binding and the high affinity of oxy-Hb for IHP. The kinetics of oxygen and carbon monoxide binding were examined in the presence of IHP; the rate constant for the dissociation of oxygen from fully saturated oxy-Hb was doubled from about 50 sec-1 to 100 sec-1 when IHP was added to phosphate-free hemoglobin, whereas in the case of carbon monoxide, the comparable dissociation rate constant increased from 0.019 sec-1 to 0.032 sec-1. Partial photodecomposition of oxy-Hb and carbon monoxide hemoglobin in the presence and absence of IHP demonstrated that the intermediate species Hb4X3 binds ligand with rate constants of 5 x 106 m-1 sec-1 and 33 x 106 m-1 sec-1, respectively (X = oxygen), whereas the corresponding rate constants for carbon monoxide are 1.4 x 106 m-1 and 7.4 x 106 m-1 sec-1. IHP was also found to induce kinetic heterogeneity in the reaction of deoxyhemoglobin with carbon monoxide which was not present in hemoglobin free of phosphates. This heterogeneity is most prominent at wave lengths near the Soret isosbestic region of the deoxy and carbon monoxide spectra and presumably reflects phosphate-induced kinetic differences between the α and β chains of hemoglobin.