E. Schnabel, C. Li
Jul 1, 1960
Citations
0
Influential Citations
18
Citations
Journal
The Journal of biological chemistry
Abstract
Recent work (l-3) on the synthesis of peptides composed of amino acid sequences that appear in both the adrenocorticotropins and the melanotropins has revealed that melanocyte-stimulating activity is enhanced as the length of the peptide chain increases (4). It was shown that the hexapeptide n-glutamyl-nhistidyl-~-phenylalanyl-~-arginyl-~-tryptophyl-glycine possesses an activity almost 10 times higher than that of the pentapeptide from which the NHz-terminal glutamyl residue is absent (‘2). The question arose whether this increase in biological activity is due specifically to the presence of the glutamyl residue itself or merely to the added length of peptide chain. We decided, therefore, to synthesize the hexapeptide with glycine at the N-terminus instead of glutamic acid. It will be seen herein that this substitution results in no alteration of melanocyte-stimulating activity. The synthesis of glycyl-n-histidyl-n-phenylalanyl-n-arginyl-b tryptophyl-glycine was accomplished by coupling the tripeptide derivative, G-tosyl-L-arginyl-n-tryptophyl-glycine methyl ester (5), with another tripeptide derivative, carbobenzoxyglycyl-nhistidyl-L-phenylalanine. The latter was obtained from the reaction of carbobenzoxyglycyl-L-histidine azide (6) with the methyl ester of n-phenylalanine (7). The azide coupling was not satisfactory because of poor solubility, and the yield was low; however, when the tripeptide ester was saponified, the derivative desired for the final coupling was obtained. The blocked hexapeptide did not crystallize; saponification yielded an amorphous product, carbobenzoxyglycyl-~-histidyl-~-phenylalanyl-~-arginyln-tryptophyl-glycine, which was then subjected to reduction with sodium in liquid ammonia (8). The free hexapeptide was desalted on an Amberlite IRC-50 (XE-64) ion exchange column, as previously described (9), and lyophylized. The content of tryptophan in the peptide was estimated spectrophotometrically (lo), and the other amino acids were determined by the method of Levy (11) ; these analyses gave molar ratios for glycine-histidine-phenylalanine-arginine-tryptophan of 2.0: 1.1:0.8:0.9: 1.0. Chymotryptic digestion of the peptide gave rise to three fragments which were identified as glycylhistidyl-phenylalanine, arginyl-tryptophan, and glycine, whereas digestion with trypsin liberated two fragments, glycyl-histidylphenylalanyl-arginine and tryptophyl-g1ycine.l The enzymic digestions were carried out at 35” for 24 hours with an enzymesubstrate ratio of 1: 100 in a solution of pH 9; the separation and