Kinetics of Oxygen Binding to Human Hemoglobin: TEMPERATURE JUMP RELAXATION STUDIES

Abstract

Abstract The kinetics of oxygen binding to human hemoglobin at pH 7, 0.1 m phosphate, 20°, 2.5 x 10-5 to 10-3 m heme has been investigated by chemical relaxation methods in order to obtain information about the elementary steps and the mechanism of the cooperative process of ligand binding. The temperature jump relaxation method allows the oxygen-binding reaction to be followed over its entire time range. The relaxation process is characterized by two phases which are well separated in time. Both the time constants and the relative amplitudes are dependent on protein and ligand concentration. The two relaxation phases can be understood qualitatively. The fast phase corresponds at low oxygen saturation mainly to the binding of the first ligand molecule and at high saturation mainly to the binding of the last ligand molecule. At intermediate oxygen saturation the fast phase is determined predominantly by the kinetics of both the first and the last step—the intermediate reaction steps do not contribute significantly. The slow relaxation phase involves all elementary binding reactions, but is determined mainly by the kinetics of the intermediate oxygen-binding steps. The relative amplitudes of the fast and the slow relaxation phases reflect strongly the population of the reaction species involved. It is concluded that hemoglobin species with one and three ligands bound must be populated measurably at equilibrium. Reaction models which assume negligible concentrations of these intermediates are therefore not valid. Three reactions have been characterized directly by their kinetic properties: (a) a fast reaction step at low oxygen saturation yielding an apparent off-rate constant of about 1000 s-1 and an apparent on-rate constant (per tetramer) of about 4 x 107 m-1 s-1; (b) a fast reaction step at high oxygen saturation, yielding an apparent on-rate constant of about 4 x 107 m-1 s-1; (c) a slow reaction, which at high oxygen concentration yields an apparent rate constant of 5 x 106 m-1 s-1. It thus appears that the binding of the first oxygen molecule to deoxyhemoglobin is a very rapid process which is characterized by an apparent rate constant as large as that found for the binding of the last oxygen molecule. The low oxygen affinity of deoxyhemoglobin arises mainly from the high dissociation rate constant, i.e. short lifetime, of Hb4O2. It cannot be decided if these kinetic properties apply equally to both the α and β chains or if only one type of chain is involved in the fast phase of the relaxation spectrum. A four-step binding model (Adair scheme) explains quantitatively the principal observations of the relaxation kinetics. Adair rate parameters have been obtained which describe the appearance of two relaxation phases only, the ligand concentration dependence (from 0 to 250 µm O2), the protein concentration dependence (from 2 x 10-5 to 10-3 m heme), and the relative and absolute relaxation amplitudes of both phases. The calculated stopped flow kinetic progress curve, using these values, is in agreement with the available data (which are in the saturation range greater than 50%) and predicts a biphasic time course, in contrast to the kinetics of CO-binding. A decision between mechanisms based upon the relaxation kinetics data can be made only with certain assumptions. The values of the Adair-recombination rate constants exclude a mechanism in which there are two very rapidly interconverting structure forms of the protein, each exhibiting equal or nearly equal intrinsic kinetic and equilibrium properties for the four subunits. A concerted mechanism might apply if structure changes are not very fast compared to ligand binding or, if the kinetic properties of the α and β chains are markedly different. However, no consistent fit of the relaxation spectrum has been obtained so far with these assumptions.