Characterization of intermediate hemoglobin produced during methemoglobin reduction by ascorbic acid.

Methemoglobin reduction by ascorbic acid was found apparently to cease halfway without further reduction. Studies by isoelectric focusing on Ampholine plate gel revealed that the solutions of the halfway reduced methemoglobin are composed of about 6% oxyhemoglobin, 59% intermediate hemoglobin, and 35% methemoglobin. The intermediate hemoglobin was isolated by CM Sephadex C-50 column chromatography and identified as alpha3+beta2+ valency hybrid by studies using the pattern of isoelectric focusing of p-chloromercuribenzoate-treated intermediate hemoglobin on Ampholine plate gel, absorption spectra, and difference spectra induced by the addition of inositol hexaphosphate in comparison with the reconstituted valency hybrids, alpha3+beta2+ and alpha2+beta3+. Essentially no alpha2+beta3+ valency hybrid was included in the intermediate hemoglobin solutions. These results suggest that methemoglobin reduction by ascorbic acid is mainly initiated by the attack of beta-methemoglobin chains accompanied by the following scheme. Methemoglobin leads to alpha3+beta2+ valency hybrid leads to oxyhemoglobin. The course of methemoglobin reduction by ascorbic acid through alpha2+beta3+ is likely to be small.