Oxidation of Carbonyl

Abstract

Oxidation reactions of carbonyl species of hemoglobins A, Zurich, and Osler with ferricyanide to form met‐hemoglobins have been investigated at pH 7.0 and 25°C. The kinetics support a two‐step process in which, first, CO is lost to give the unliganded reduced species (deoxy), followed by oxidation in a one‐electron transfer to ferricyanide ion. On and off rates for CO markedly affect the overall oxidation rate. That α subunits oxidize more rapidly than B subunits was noted by changes in C–O stretch frequency during the course of reactions with Hb A and Hb Zurich. The substitution of Arg for His at β63 in Hb Zurich resulted in a slower overall oxidation rate ascribable to greater affinity for CO. MetHb formation from stripped HbCO A was biphasic with the rate for the final ∼ 50% of the reaction about 30% faster than that for the initial ∼ 50%. Addition of inositol hexakis‐phosphate increased rates several‐fold in both phases and shifted the point of change from slow to fast phase. These effects suggest that inositol hexakisphosphate binds weakly (Kd∼ 30 μM) to Hb4(CO)4 in a high‐affinity state which, after loss of one CO, changes to a low‐affinity state where inositol hexakisphosphate binds more strongly (Kd < 1 μM) for the remainder of the reaction. With Hb Osler, which exhibits very low heme‐heme interaction and reacts poorly with inositol hexakisphosphate, the reaction was monophasic with a rate throughout comparable to the initial rate for stripped HbCO A. The very low level of CO saturation (< 1–6%) usually found in vivo with normal hemoglobin subunits should not greatly affect overall rates of methemoglobin formation from one‐electron electron acceptors. However, with the high levels of CO found bound (20–100% saturation) to the β subunit of hemoglobin Zurich in blood, the exposure to CO (as in smoking) can diminish the extent to which the abnormal Zurich subunit will be oxidized. CO exposure may, therefore, reduce significantly some of the undesired consequences of having hemoglobin Zurich in red blood cells.