The oxidation of cat, human, and the cat-human hybrid hemoglobins alpha 2 human beta 2 cat and alpha 2 cat beta 2 human by copper(II).

ABSTRACT

The heme iron of the beta chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the beta globin chain. The presence of freely reactive -SH groups on the alpha chains of cat and sheep hemoglobins does not alter the course of this reaction; only the beta hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two beta chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, alpha 2 Human beta 2 Cat and alpha 2 Cat beta 2 Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the beta subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid alpha 2 Cat beta 2 Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or alpha 2 Human beta 2 Cat. Cat beta globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2 beta. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the beta chain and is associated with histidine 2 beta.