Quantitative determination of amino acid racemization in heat-alkali-treated melanotropins: Implications for peptide hormone structure-function studies

The treatment of frog skins (in vitro) and frogs (in vivo) with melanotropins that have been heated briefly in aqueous alkali resulted in prolonged skin darkening. It has been postulated that this increase in melanotropic activity is related to the partial racemization of amino acid residues of the melanotropins. Quantitative determination of the extent of racemization of eight amino acids (Val, Pro, Met, Phe, Glu, Asp, Nle, Ser) present in α-melanotropin (α-MSH), [4-norleucine]-α-MSH, β_porcine-melanotropin (β_p-MSH), and [7-norleucine]-β_p-MSH after brief heat-alkali treatment, was accomplished using a high-resolution gas chromatographic technique. Phenylalanine-7 in α-MSH and [4-norleucine]-α-MSH and phenylalanine-10 in β_p-MSH and [7-norleucine]-β_p-MSH were found to be partially racemized to a greater extent than expected. Other amino acid residues were also racemized to unexpected degrees. The subsequent synthesis of an α-MSH analog containing d-phenylalanine-7, [4-norleucine, 7-d-phenylalanine]-α-MSH, resulted in a highly potent melanotropin with ultralong biological activity, as determined by frog skin bioassay, stimulation of mouse melanoma cell tyrosinase activity, and activation of mouse melanoma adenylate cyclase.