A NOVEL METHOD FOR THE AMINO-ACID-SEQUENCE CONFIGURATION DETERMINATION OF PEPTIDES CONTAINING D L-AMINO ACIDS UTILIZING A FLUOROGENIC EDMANREAGENT, OSULPHONYL-4-(2,1,3-BENZOXADIAZOLYL)ISOTHIOCYANATE (DBD-NCS)/

A novel method for the amino acid sequence and configuration determina tion of peptides containing D- or L-amino acids is presented. An enkep halin analogue, [D-Ala(2), D-Leu(5)]enkephalin (Tyr-Ala-Gly-Phe-Leu) w as derivatized with a fluorogenic Edman reagent, N-dimethylaminosulpho nyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS), cleaved and cy clized with trifluoroacetic acid at 50 degrees C for 1 min and the res ultant thiazolinone derivative (DBD-thiazolinyl-L-Tyr) was separated f rom the racemized DBD-thiazolinyl-D-Tyr (about 20% as compared to the L-isomer) on a phenylcarbamylated cyclodextrin column: The separation factor (alpha) for D- and L-isomers was 1.09. The column eluate was mo nitored fluorometrically at 524 nm with excitation at 387 nm. The dete ction limit was about pmol range. The same treatment adopted for the r esidual peptide, Ala-Gly-Phe-Leu, gave DBD-thiazolinyl-D-Ala (alpha = 1.09 for D,L-Phe) with the lesser amount of L-analogue (about 20%). In the same manner, Gly and L-Phe (alpha = 1.09) were detected. The meth od might be useful for the study of aging of proteins such as eye lens and the amyloid proteins derived from Alzheimer's disease.