Mechanism of methemoglobin reduction by ascorbic acid under anaerobic conditions.

The time course of methemoglobin reduction by ascorbic acid under anaerobic conditions was analyzed by using isoelectric focusing on Ampholine plate gel in order to compare results obtained by studies of the changes in absorption during the reaction. The intermediate hemoglobin which appeared all through the reaction was single and identified as the alpha3+beta2+ valency hybrid. In the presence of inositol hexaphosphate, reduction of methemoglobin was considerably accelerated and this acceleration was restricted to the step in which methemoglobin is reduced to the alpha3+beta2+ valency hybrid. The phase containing alpha3+beta2+ valency hybrid reduction to deoxyhemoglobin was not affected by the presence of this organic phosphate. The reaction rate constant of each phase was estimated by the analysis of the changes in three components such as methemoglobin, alpha3+beta2+ valency hybrid, and deoxyhemoglobin. The specific attack of beta-methemoglobin chains in methemoglobin tetramer by ascorbic acid was explained by the functional differences in alpha- and beta-methemoglobin chains in the protein.