Studies on the Intermediates of Methemoglobin by the Use of a Cross-Linking Agent

Abstract

The intermediates of methemoglobin [MetHb] were investigated by the use of an intramolecular cross-linking agent, bis(3, 5-dibromosalicyl)fumarate. The cross-linked Hb A [HbFu], which cannot split into αβ-dimers, was oxidized with potassium ferricyanide, sodium nitrite and seasamol. In every oxidation, cross-linked hemoglobins with 1-met, 2-met, 3-met, and 4-met [MeHbFu] subunits were formed. As the oxidation level increased, the contents of the hemoglobins with higher amounts of met subunits increased. MetHbFu may be produced via the hemoglobins with 1-met→2-met→3-met subunits. The hemoglobins with 1-met and 3-met subunits may each contain two valency asymmetrical hybrids. Whereas analysis by isoelectrofocusing showed that Hb A partially oxidized with potassium ferricyanide contained Hb A, α⁺₂β₂, α₂β⁺₂ and MetHb, all being symmetrical hybrids, treatment of the oxidized Hb A with the cross-linking agent gave corss-linked hemoglobins with 3-met subunits (valency asymmetrical hybrids) besides those with 2-met and 4-met subunits. In the course of oxidation of native Hb A, unstable valency asymmetrical hybrids may be initially produced but may split into the αβ-dimers which recombine and form stable valency symmetrical hybrids under analysis.