Autoxidation of human hemoglobin: kinetic analysis of the pH-profile

The autoxidation reaction of human hemoglobin A was studied over the pH range of 5.3-10.4 in 0.1 M buffer at 37 degrees C. In the first-order plot, hemoglobin showed a biphasic reaction, which consisted of a rapid initial reaction and was followed by a slower second phase in the acidic or neutral pH range (5.3-8). However, this difference decreased with increase of pH of the solution and only a single-phase reaction was observed in alkali solutions above pH 8. For the kinetic analysis of the time course of the reaction, computer fitting was made to obtain curves as a function of time by a non-linear least squares method using a biphasic first-order equation with rate constants of kf and ks for the initial fast component reaction and for the slower second reaction, respectively. The parameter of the molar fraction (P) with a rate constant of kf was computed in the range of 0.45-0.55, which means that hemoglobin was autoxidized with half of the component via the reaction process with the initial rapid rate (kf) and the other half via the reaction with the following slower rate (ks). kf and ks thus obtained versus pH plot also indicated that (1) both rate constants increase markedly with increase in hydrogen ion concentrations, but (2) when pH > 8, kf becomes equal to ks with P = 1.0, and (3) there appears a rate minimum at pH 8-8.5, followed by a considerable elevation of the rate with increase in hydroxyl ion concentration. In order to examine these complicated pH-profiles, some mechanistic models were proposed for the autoxidation reaction of hemoglobin. Fitting of their rate equations as a function of pH was examined for the experimental rate constants kf and ks versus pH plot by the least squares method with use of a computer. Both pH-profiles could be best explained by the "acid-base catalyzed model." This revealed not only the catalytic role of hydrogen ions but also the involvement of hydroxyl ions and water molecules in the autoxidation reaction of human hemoglobin.