Kinetics of the Reaction with Oxygen of Mixtures of Oxy- and Carbon Monoxide Hemoglobin

The paper reports rapid mixing and relaxation experiments performed on mixtures of oxy- (HbO2) and carbon monoxide (HbCO) human hemoglobin. On the (well justified) assumption that the two ligands will distribute at random between the available sites, intermediates containing different proportions of O2 and CO will be formed. In the stopped flow experiments mixtures containing different proportions of the two ligands have been mixed with sodium dithionite (Na2S2O4), which rapidly reduces to zero the O2 concentration in the system. The apparent dissociation velocity constant for O2 (koff) measured under these conditions decreases progressively as the fraction of HbCO in the mixture increases, in agreement with previous observations on sheep hemoglobin. Temperature jump experiments performed on mixtures of HbCO and HbO2 show that the amplitude of the faster relaxation time (τf) relative to that of the slower one (τf) increases as the percentage of HbCO in the mixture is progressively increased. At high enough percentage of HbCO (≥70%), the amplitude of the faster relaxation time becomes dominant. The reciprocal relaxation time (τf-1), measured under these conditions, is linearly dependent on oxygen concentration, while it is independent of protein concentration (so long as O2 is buffered). The apparent second order velocity constant is kon = 4.8 x 107 m-1 s-1 at 25°. Simple considerations indicate that the kinetics of the reaction with oxygen of mixtures containing high enough percentages of HbCO should represent the combination and dissociation velocity constants of high affinity forms of hemoglobin.