A NOVEL METHOD FOR THE AMINO-ACID-SEQUENCE CONFIGURATION DETERMINATION OF PEPTIDES CONTAINING D/L-AMINO ACIDS UTILIZING A FLUOROGENIC EDMAN REAGENT, 7-N,N-DIMETHYLAMINOSULPHONYL-4-(2,1,3-BENZOXADIAZOLYL)ISOTHIOCYANATE (DBD-NCS)

A novel method for the amino acid sequence and configuration determination of peptides containing D- or L-amino acids is presented. An enkephalin analogue, [D-Ala(2), D-Leu(5)]enkephalin (Tyr-Ala-Gly-Phe-Leu) was derivatized with a fluorogenic Edman reagent, 7-N,N-dimethylaminosulphonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS), cleaved and cyclized with trifluoroacetic acid at 50 degrees C for 1 min and the resultant thiazolinone derivative (DBD-thiazolinyl-L-Tyr) was separated from 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 monitored fluorometrically at 524 nm with excitation at 387 nm. The detection limit was about pmol range. The same treatment adopted for the residual 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 method might be useful for the study of aging of proteins such as eye lens and the amyloid proteins derived from Alzheimer's disease.