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

Abstract

The recently discovered native endomorphins play an important role in opioid analgesia, but their metabolic fate in the organism remains relatively little 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 endomorphins with proteolytic enzymes. The native endomorphin-1, H–Tyr–Pro–Trp–Phe–NH 2 (1), and endomorphin-2, H–Tyr–Pro–Phe–Phe–NH 2 (2), and an analog of endomorphin-2, H–Tyr–Pro–Phe–Phe–OH (3), were synthetized, and the levels of their resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptidase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyzes the C-terminal amide group to a carboxy group, and then splits the peptides at the Trp 3–Phe 4 or Phe 3–Phe 4 bond. The remaining fragment peptides are stable against the enzymes investigated. Carboxypeptidase A degrades only analog 3 at the Phe 3–Phe 4 bond. Aminopeptidase M cleaves the peptides at the Pro 2–Trp 3 or Pro 2–Phe 3 bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe–NH 2-s while the N-terminal part displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide group is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side.