The kinetics of dissociation of the first oxygen molecule from fully saturated oxyhaemoglobin in sheep blood solutions

A detailed description is given of a stopped-flow apparatus, which is specially suitable for measuring the rates of the rapid reactions of haemoglobin with oxygen and carbon monoxide. The apparatus has been chiefly applied to determinations of the rate of displacement of oxygen, r, from combination with haemoglobin by carbon monoxide. It is shown theoretically, and confirmed experimentally, that the reciprocal of r when plotted against the oxygen pressure initially in equilibrium with the haemoglobin gives a straight line from which k4, the velocity constant of the reaction Hb4O8→Hb4O6+O2, can be readily calculated. The value of k4 is scarcely affected by pH over the range 9.1 to 7.1, but has a temperature coefficient at both pH values of about 3.2 per 10° C. Measurements have also been made of the overall velocity constants of combination of oxygen (k') and of carbon monoxide (l') with sheep haemoglobin, and of the overall rate of dissociation of sheep oxyhaemoglobin in presence of Na2S2O4 (k). The numerical values tally well with those obtained by previous rougher methods. The effect of p-chloromercuribenzoate (PCMB) on k4, k', l' and k has been determined. This reagent has only a slight effect on the combination velocity constants k' and l', but increases markedly the value of k4. Its effect on k is very complicated, suggesting that the velocity constants of the reactions Hb4O6→Hb4O4+O2 and Hb4O4→Hb4O2+O2 may be increased, whereas that of the reaction Hb4O2→Hb4+O2 may be greatly reduced. These curious effects are qualitatively in line with the effects of PCMB on the equilibrium between oxygen and haemoglobin.