Hemoglobin autoxidation and regulation of endogenous H 2O 2 levels in erythrocytes

Abstract

Red cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H 2O 2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H 2O 2 and superoxide in the erythrocyte were used to estimate H 2O 2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 × 10 −7 s −1 and an endogenous H 2O 2 and superoxide concentrations of 5 × 10 −11 and 5 × 10 −13 M, respectively, lower than previous estimates.