Liquid chromatographic study of the enzymatic degradation of endomorphins,with identification by electrospray ionization mass spectrometry

The recently discovered native endomorphins play an important role in opioi d analgesia, but their metabolic fate in the organism remains relatively li ttle known. This paper describes the application of high-performance liquid chromatography combined with electrospray ionization mass spectrometry to identify the degradation products resulting from the incubation of endomorp hins with proteolytic enzymes. The native endomorphin-1, H-Tyr-Pro-Trp-Phe- NH, (1), and endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2 (2), and an analog of end omorphin-2, H-Tyr-Pro-Phe-Phe-OH (3), were synthetized, and the levels of t heir resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptid ase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyz es the C-terminal amide group to a casboxy group, and then splits the pepti des at the Trp(3)-Phe(4) or Phe(3)-Phe(4) bond. The remaining fragment pept ides are stable against the enzymes investigated. Carboxypeptidase A degrad es only analog 3 at the Phe(3)-Phe(4) bond. Aminopeptidase M cleaves the pe ptides at the Pro(2)-Trp(3) or Pro(2)-Phe(3) bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe-NH2-s while the N-terminal p art displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide gr oup is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side. (C) 1999 Elsevier Science B.V. All rights re served.