Interactions of rat red blood cell sulfhydryls with arsenate and arsenite

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Abstract
Arsenic—thiol interactions were investigated by determining changes in rat blood sulfhydryls after exposure to arsenate, As(V), or arsenite, As(III). Incubation with As(V) resulted in time‐and dose‐dependent depletion of nonprotein sulfhydryls (NPSH), specifically glutathione (GSH). At the highest As(V) concentration (10 mM), significant loss of glutathione was only observed after 3 h of incubation, but by 5 h 0.5 mM As(V) and higher was sufficient to deplete CSH. As(V) was reduced to As(III) at all dose levels, indicating a redox interaction with GSH, but oxidized glutathione (GSSG) was not formed in sufficient quantities to account for losses in GSH. This may be due to formation of another oxidized species such as a protein‐mixed‐disulfide (ProSSG). Further evidence that glutathione reduces arsenate was obtained by pretreating cells with the sulfhydryl derivatizing agent N‐ethylmaleimide (NEM). Removal of thiols with NEM severely inhibited the formation of As(III) in these incubations, indicating that the main pathway for arsenate reduction in red cells is sulfhydryl dependent. As(III) demonstrated a completely different profile of sulfhydryl interaction. Sulfhydryls (NPSH and GSH) were depleted but the losses were primarily accounted for by oxidation to CSSC. As(III) was also a more potent sulfhydryl depleting agent, requiring only 0.1 mM As(III) to significantly reduce GSH after 5 h of incubation. Significant levels of GSSG formed at all doses of As(III). Evidence is presented to suggest that As(III) also formed mixed complexes with protein and glutathione. Samples that were acid precipitated displayed loss of cytosolic glutathione, which could be reversed if NEM was added prior to protein precipitation. Arsenic was detected in high quantities in the protein precipitates, and this was also found to be reversible by NEM treatment. The fact that both GSH depletion and protein binding were reversible by NEM treatment points to formation of a mixed complex of protein, GSH, and As(III), possibly ProS‐As‐(SG)x. Arsenic affinity chromatography and polyacrylamide gel electrophoresis were used to characterize arsenic binding proteins in red‐cell cytosol. The main arsenic binding protein appeared to be hemoglobin.