Kinetics of heme binding to semi-alpha-hemoglobin.

The binding of carbonmonoxyheme to semi-alpha-hemoglobin and to an apohemoglobin control was investigated using stopped-flow techniques in 0.025 M potassium phosphate buffer, pH 7 and 10 degrees C. The resultant second order kinetic data were analyzed by the classical model which assumes the existence of an intermediate complex which either redissociates to reactants or undergoes an irreversible conversion to form hemoglobin. The rate constants for the latter unimolecular process were apparently not experimentally different for semi-alpha-hemoglobin and apohemoglobin (360 ( +/- 100) s-1 and 480 ( +/- 60) s-1, respectively). However, the equilibrium dissociation constant for the intermediate of semi-alpha-hemoglobin (Kd = 9.3 ( +/- 2.6) micromolar) was approximately two fold greater than that of apohemoglobin (Kd = 4.1 ( +/- 0.5) micromolar). The reduced stability of the semi-alpha-hemoglobin complex was postulated to be due to the lower affinity of the beta pocket for heme. The studies reported here address the possible role of semi-alpha-hemoglobin as an intermediate in the assembly of hemoglobin in vivo.