Assay for Enkephalin-Generating Enzyme in Rat Brain Tissues by High Performance Liquid Chromatography with Postcolumn Fluorescence Derivatization

Abstract

A simple and sensitive method for assaying enkephalin-generating enzyme activity in rat brain tissues is described. Methionine-enkephalin, produced enzymatically from α-endorphin and BAM-12P as substrates and leucine-enkephalin from α-neoendorphin, were quantified by high performance liquid chromatography with postcolumn fluorescence derivatization using hydroxylamine, cobalt(II) and borate. The detection limits for methionine- and leucine-enkephalin- generating activities of the enzyme are 2.5 and 3.0pmol min^-1 mg-protein^-1, respectively. The enzyme can be characterized as an endo-oligopeptidase A-like enzyme on the basis of inhibition studies.