Reversal of copper(II)-induced methemoglobin formation by thiols

Oxyhemoglobin is oxidized to methemoglobin by copper(II) in a two-stage reaction that results in conversion of Fe(II) to Fe(III) in heme of the beta subunits, but not the alpha subunits. Glutathione and other thiols, which are slowly oxidized by Cu(II), protect oxyhemoglobin from Cu(II)-induced oxidation. In the present studies, when oxyhemoglobin was first oxidized to methemoglobin by Cu(II) and thiols such as glutathione added to the sample, methemoglobin was reduced to oxyhemoglobin. Once reduction of methemoglobin stopped, as the thiol was oxidized, the oxyhemoglobin formed was reoxidized by Cu(II). The addition of the same thiols to methemoglobin formed by autoxidation did not reduce it to oxyhemoglobin. The addition of thiols such as cysteine, which are rapidly oxidized by Cu(II), to methemoglobin formed by incubation with Cu(II) also resulted in reduction of methemoglobin, but the period of reversal was much shorter than that seen with glutathione and other less reactive thiols. When cysteine and glutathione were added together to Cu(II)-induced methemoglobin, the rate of reduction and reoxidation was intermediate to that seen when either was added separately. When EDTA was added to a system in which oxyhemoglobin was undergoing Cu(II)-induced oxidation, oxidation of oxyhemoglobin ceased and there was no reduction of the methemoglobin to oxyhemoglobin. When both glutathione and EDTA were added to this system, the response was the same as with EDTA alone, suggesting that Cu(I) or (II) may be required for the reduction of copper-induced methemoglobin by thiols. These studies show that thiols that are slowly oxidized by Cu(II) both protect oxyhemoglobin from Cu(II)-induced oxidation, and reduce the methemoglobin formed to oxyhemoglobin.