DETERMINATION OF DISULFIDE STRUCTURE IN AGOUTI-RELATED PROTEIN (AGRP)BY STEPWISE REDUCTION AND ALKYLATION

The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide c hain of 112 amino acid residues, consisting of an N-terminal acidic re gion and a unique C-terminal cysteine-rich domain. The disulfide struc ture of recombinant human AGRP was determined by chemical methods usin g partial reduction with tris(2-carboxyethyl)phosphine under acidic co nditions, followed by direct alkylation with N-ethylmaleimide or fluor escein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible dis ulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C8 8, C67-C82, and C81-C99, respectively. The chemically defined disulfid e connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.