Molecular Pharmacology, Vol 11, 326-334, Copyright © 1975 by the American Society for Pharmacology and Experimental Therapeutics

Kinetics of Ferrihemoglobin Formation by Some Reducing Agents, and the Role of Hydrogen Peroxide

¹ Pharmakologisches Institut der Universität München, Munich, German Federal Republic

The rate of oxidation by hydrogen peroxide of human hemoglobin, virtually free from catalase, glutathione peroxidase, and superoxide dismutase, was found to be proportional to the concentrations of hemoglobin and hydrogen peroxide, the second-order rate constant at pH 7.4 and 37° being k = 125 M^-1 sec^-1. Formation of ferrihemoglobin by reduced glutathione in air was found to be slow, gaining its maximal velocity after a lag phase. Kinetic data and the effect of catalase or glutathione peroxidase demonstrated that hydrogen peroxide is an essential intermediate which produces ferrihemoglobin in solutions of hemoglobin and reduced glutathione. The much higher rate of ferrihemoglobin formation by phenylhydroxylamine than by hydrogen peroxide and the failure of catalase to inhibit the reaction showed that hydrogen peroxide is not an important intermediate in the formation of ferrihemoglobin by phenylhydroxylamine. The reaction rate was found to be proportional to the concentrations of phenylhydroxylamine and hemoglobin. The second-order rate constant was calculated to be k = 2350 M^-1 sec^-1. With the formation of ferrihemoglobin by 4-dimethylaminophenol also, reaction rates and the failure of catalase to inhibit the reaction demonstrated that hydrogen peroxide is of no importance. The lag phase of the reaction suggests that oxidation products of 4-dimethylaminophenol produced by the reaction between oxyhemoglobin and 4-dimethylaminophenol are essential intermediates.