Abstract: | A convenient procedure was developed for identifying selenocysteine in selenoproteins by mass spectroscopy, based on formation of the 2,4-dinitrophenyl (DNP) derivative. Pure ovine erythrocyte glutathione peroxidase was reduced with sodium borohydride and reacted with 1-fluoro-2,4-dinitrobenzene at neutral pH under anaerobic conditions in 4 M guanidine. The inactivated enzyme was hydrolyzed with 6 N HCl for 20 h at 110 degrees C under anaerobic conditions. Following extraction of the hydrolysate with benzene, Se-(2,4-dinitrophenyl)selenocysteine in the aqueous phase was separated from non-DNP-amino acids by gel-filtration chromatography and then separated from other water-soluble DNP-amino acids by reversed-phase high-performance liquid chromatography. The Se-(2,4-dinitrophenyl)selenocysteine was converted to Se-methyl-N-(2,4-dinitrophenyl)selenocysteine by the addition of sodium barbital to induce an intramolecular Se leads to N shift (Smiles rearrangement) under anaerobic conditions, in the presence of methyl iodide to trap the liberated selenol group. Following esterification of the product's carboxyl group with methanol and hydrochloric acid, it was subjected to direct probe mass spectroscopy and identified as the methyl ester of Se-methyl-N-(2,4-dinitrophenyl)selenocysteine. This procedure allows selenocysteine to be isolated quite easily as a readily identifiable derivative and has permitted the first identification of a seleno amino acid in a protein by mass spectroscopy. |