Mechanism of Autoxidation of Hemoglobin Kempsey (β99 ASP→ASN)

Abstract

The mechanism of autoxidation of oxyhemoglobin Kempsey (β99 asp → asn) was studied at pH 7.0, 37°C, using isoelectric focusing electrophoresis. During autoxidation, two intermediate hemoglobins, (α²⁺β³⁺)₂ and (α³⁺β²⁺)₂ were observed, and these were consecutively changed to methemoglobin. The autoxidation rates of oxyhemoglobin Kempsey were reduced about 43% by the addition of catalase and superoxide dismutase, but were not significantly changed by the addition of inositol hexaphosphate. This abnormal hemoglobin autoxidized more slowly than normal hemoglobin A. The differences in the autoxidation rates between these hemoglobins were explained by the changes in quaternary structure of these proteins. The reaction rate constant, k+1, k+2, k+3 and k+4 of each step including the reactions: were determined by the analysis of fractional changes in these hemoglobin derivatives during the autoxidation. The difference in the reaction rate constants between k+1 and k+3 was explained by the nonequivalence of α and β chains in tetrameric oxyhemoglobin Kempsey. The reaction mechanism of autoxidation of hemoglobin Kempsey was discussed on the basis of these kinetic results.