| Summary: | Inorganic arsenic administered to animals is primarily associated with liver cytosolic macromolecules. The specifics of this binding and its role in arsenic toxicity have never been fully examined. This dissertation concentrated on determining the species of inorganic arsenic which binds to liver cytosolic proteins, the specific proteins involved and the characteristics of their arsenic binding using an in vitro incubation system to [⁷³As]-label proteins. Arsenite was the species which bound cytosolic proteins and this was attributed to its affinity for protein sulfhydryls. Ammonium sulfate precipitation of cytosolic proteins revealed three arsenite-binding proteins (AsBPs) with molecular weights of 100K, 450K and >2000K as determined by size-exclusion chromatography. Similar-sized proteins were observed in liver cytosol prepared from a rabbit administered [⁷³As]-arsenite in vivo. The binding of arsenite to the 450K AsBP was most stable and further work focused on its purification and characterization. Trivalent arsenic affinity chromatography suggested a dithiol-like binding site. Amino acid composition indicated less than 0.5 mol% cysteine residues and implied that other thiol-containing functional groups may be used to complex arsenite. Arsenite inhibits enzymes which contain protein-bound lipoic acid, such as pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, by presumably binding to these dithiol functional groups. Thin-layer chromatography and mass spectrometry established that arsenite complexes with reduced lipoic acid and might be binding to similar dithiol groups on the 450K AsBP. Comparison of the arsenite-binding activities of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and cytosol showed increases for the enzymes but a decrease for cytosol after pre-treatment with NADH which reduces lipoic acid groups. However, the arsenite-binding activity of the 450K AsBP increased by 26-fold. Antibody affinity studies, though, showed that the 450K AsBP was not recognized by antibodies which cross-react with lipoic acid epitopes on proteins. Preparative SDS-PAGE was successful in purifying the 450K AsBP by over 2300-fold. Attempts at sequencing this protein indicated a blocked N-terminus. Chemical cleavage by cyanogen bromide produced two fragments of approximately 60K and 53K. Isolation of these fragments and their sequencing is continuing.
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