Micro-scale sequence analysis from the N-terminus of peptides using the fluorogenic Edman reagent...

The fluorogenic Edman reagent 4-N,N-dimethylamino-1-naphthyl isothiocyanate (DNTC) was reacted with amino acids and peptides, cyclized by acid and the liberated 4-N,N-dimethylamino-1-naphthyl thiohydantoin (DNTH) amino acids were then separated and detected by HPLC. The fluorescence intensities of DNTH-amino acids except DNTH-proline and -serine were dramatically increased in the alkaline solution and organic solvent. Thus, the postcolumn reaction with alkaline acetonitrile solution was adopted in HPLC. The polar and aromatic amino acids afforded two DNTH-amino acids on derivatization with DNTC and cyclization with acids. These were suggested to be stereoisomers of DNTH-amino acids. The sequence analysis of 0.5 nmol Leu-enkephalin was achieved by the double coupling method with DNTC and phenyl isothiocyanate followed by the proposed HPLC system.