Influence of glutathione on the formation of cysteine alkylation products in human hemoglobin

Abstract Human blood samples were treated in vitro with iodoacetamide. At low concentrations - less than 1 mM - only a low fraction of the β93 cysteine in hemoglobin was alkylated, whereas the alkylating reaction with glutathione was extensive. At higher iodoacetamide concentrations the glutathione pool became exhausted leading to more than proportional increases in the alkylation of the sulfhydryl group in hemoglobin. When diethyl maleate was used as a glutathione depletor prior to incubation with iodoacetamide, low concentrations of iodoacetamide were sufficient to obtain high degrees of hemoglobin sulfhydryl alkylation. N -Ethylmaleimide could not be used as glutathione depletor because the reaction with glutathione appeared to be reversible. The lower reactivity of the thiol group in hemoglobin in comparison with that of glutathione was also found for the isolated biomolecules. The protection of hemoglobin by glutathione present in the human erythrocyte renders the measurement of hemoglobin alkylation less attractive for biological effect monitoring. The sensitivity of such methods is lowered, while the important relation between the alkylation of hemoglobin and that of DNA in the target tissues is affected by interindividual differences in the ratios of the effectiveness of glutathione protection between erythrocytes and target cells.