The kinetics of deoxygenation of human haemoglobin

Abstract

Spectrokinetic studies of the deoxygenation of human haemoglobin in the presence of sodium dithionite have been made under various conditions with a constant-flow rapid-reaction apparatus and a photoelectric spectrophotometer. A reproducibility of + 3-5% in estimates of the first-order velocity coefficient has been demonstrated. No significant differences were detected between the kinetic behaviour at several pH values of oxyhaemoglobin samples from normal individuals and from persons suffering from several types of anaemia, including sickle-cell anaemia. The greatest difference observed between two haemoglobin samples was 6% in the specific reaction rate. Significant differences between the results of analyses of the reaction mixture, by measurements of light absorption at several wavelengths, show that the deoxygenation is accompanied by secondary reactions involving the pigment. The errors thereby introduced are not large, but realization of the full benefits of the precision of the technique in the interpretation of the detailed time course of the reaction is restricted. The time course of the overall reaction cannot be accurately described by the simple first-order relation, d[HbO2]dt = k[HbO2]. The calculated value of k increases from an initial value of < 0-6ka to a constant value of ka when reaction is 40% complete. This initial trend in k is not affected by wide variations of the initial oxyhaemoglobin and dithionite concentrations, and is evident in measurements at five wavelengths at pH 8.4 and two wavelengths at pH 6.3-9.5, and is therefore considered to be a genuine kinetic feature of the deoxygenation. This confirms the conclusion of Legge and Roughton (1950) for the deoxygenation of sheep haemoglobin, and is in agreement with other recent work on the kinetics and equilibria of sheep haemoglobin and with the intermediate-compound theory. Variations of the rate of deoxygenation with pH are consistent with the presence of an oxylabile group with pK 7.0. The specific reaction rate is five times as great at pH 6.0 as at pH 8.5-9.5. There was no evidence of a pH effect on the initial upward trend in the specific reaction rate. Activation energies of 22 000 cal. at pH 6.3 and 26 000 cal. at pH 8.4 have been calculated from the variation of the rate of deoxygenation with temperature. There was no evidence that the early and late stages of the overall deoxygenation were differently affected by temperature change. With increase of ionic strength, the specific reaction rate decreases at pH values below 7.0 and increases at pH values above 7.0. The results are considered in relation to other recent work, and certain anomalies are discussed.