INTERACTIONS OF RAT RED-BLOOD-CELL SULFHYDRYLS WITH ARSENATE AND ARSENITE

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 dep letion of nonprotein sulfhydryls (NPSH), specifically glutathione (GSH ). At the highest As(V) concentration (10 mM), significant loss of glu tathione was only observed after 3 h of incubation, but by 5 h 0.5 mM As(V) and higher was sufficient to deplete GSH. As(V) was reduced to A s(III) at all dose levels, indicating a redox interaction with GSH, bu t 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 e vidence that glutathione reduces arsenate was obtained by pretreating cells with the sulfhydryl derivatizing agent N-ethylmaleimide (NEM). R emoval of thiols with NEM severely inhibited the formation of As(III) in these incubations, indicating that the main pathway for arsenate re duction in red cells is sulfhydryl dependent. As(III) demonstrated a c ompletely different profile of sulfhydryl interaction. Sulfhydryls (NP SH and GSH) were depleted but the losses were primarily accounted for by oxidation GSSG. As(III) was also a more potent sulfhydryl depleting agent, requiring only 0.1 mM As(III) to significantly reduce GSH afte r 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 mi xed complexes with protein and glutathione. Samples that were acid pre cipitated displayed loss of cytosolic glutathione, which could be reve rsed if NEM was added prior to protein precipitation. Arsenic was dete cted in high quantities in the protein precipitates, and this was also found to be reversible by NEM treatment. The fact that both GSH deple tion and protein binding were reversible by NEM treatment points to fo rmation of a mixed complex or protein, GSH, and As(III), possibly ProS -As-(SG)(x). Arsenic affinity chromatography and polyacrylamide gel el ectrophoresis were used to characterize arsenic binding proteins in re d-cell cytosol. Tile main arsenic binding protein appeared to be hemog lobin.