The basis for EDTA-stimulation of methemoglobin reduction in hemolysates of human erythrocytes.

THE BASIS FOR EDTA-STIMULATION OF METHEMOGLOBIN REDUCTION IN HEMOLYSATES OF HUMAN ERYTHROCYTES Lucy Jean Sannes* and Donald E. Hultquist ** Department of Biological Chemistry The University of Michigan Ann Arbor, Michigan 48109 Received November lo,1979 Summary: We have studied the stimulation by EDTA of methemoglobin reduction in hemolysates of human erythrocytes. The EDTA effect has been shown not to be the result of an allosteric interaction of EDTA with hemoglobin or the result of a photochemical reduction. The effect does not appear to be due to a direct interaction of free EDTA with either of the catalytic components of the erythrocyte methemoglobin reduction system. The EDTA stimulation seen in hemolysates is due to the formation of an iron-EDTA complex, which transfers electrons from the reductase to methemoglobin. In a previous study (1) we observed that 0.5 mM EDTA stimulates methemoglobin reduction 60 to 70% in hemolysates of human erythrocytes. Stimulation of methemoglobin reduction by EDTA has been reported previously (2,3). There are several plausible explanations for the observed stimulations. The effect may be due to a direct binding of EDTA to methemoglobin. EDTA acts as an allosteric effector of hemoglobin (4), as do organic phosphates such as DPG*** and IHP (5). Binding of polyanions also causes conformational changes in methemoglobin (6-8) and alters its oxidation-reduction equilibria (9,101. Moreover, IHP increases the rate of methemoglobin reduction by purified erythrocyte NADH-dependent reductase in the absence (11,12) or the presence (13) of cytochrome b -5’ DPG stimulates methemoglobin reduction by ascorbic acid (14). A second possible explanation for the stimulation would be an EDTA-dependent photochemical reduction of one of the components of the methemoglobin reduction system. EDTA and flavin have been shown to be an effective system for the photoreduction of many oxidation-reduction proteins (15). Thirdly, the stimulation might be a