Isolation of Peptides Containing Solvent Accessible Histidines in Arsenite Oxidase from Alcaligenes faecalis

Abstract

Arsenite oxidase is a Molybdenum (Mo) containing hydroxylase which oxidizes arsenite (AsII 1 ) to form arsenate (Asv). Recent data (Ellis et al., 2001, McNellis and Anderson, 1998) suggest one or more histidines may be present in the active site of arsenite oxidase and serve as a binding site for arsenite. Solvent accessible histidines in arsenite oxidase were modified with 14C-diethylpyrocarbonate (DEPC) (Miles, 1977); the remaining histidines were modified with unlabeled DEPC. The large protein was cleaved into smaller peptides by digestion with trypsin. These peptides were separated by reverse phase high-pressure liquid chromatography (HPLC). The radiolabeled fractions, representing those with labeled histidines, were further purified and will be sequenced. Comparison of the peptidyl sequence to that of the X-ray crystallographic structure will indicate if histidines putatively located close to the molybdenum atom are possible binding sites for arsenite.