LIQUID-CHROMATOGRAPHY OF PEPTIDES TREATED WITH FLUOROGENIC REAGENTS AND ITS APPLICATION TO ANALYSES OF OPIOID-PEPTIDES, THEIR PRECURSORS AND RELATED ENZYMES IN RAT-BRAIN

Many opioid peptides having N-terminal tyrosyl residue of their amino acid sequence occur at low concentrations in mammalian brain tissues a nd body fluids. Their endogenous concentrations are controlled by synt hetic and metabolic enzymes. It is therefore of great importance to de velop an analytical protocol that permits facile and sensitive determi nation of various opioid peptides and related enzyme activities. Sever al chemical reactions with fluorogenic reagents have been developed fo r liquid chromatographic detection of peptides, increasing either sens itivity or selectivity. Among them, a fluorogenic chemical reaction us ing three reagents such as hydroxylamine, cobalt(II) and berate salts which provides fluorescent derivatives for only N-terminal Tyr-contain ing peptides is the most applicable to the chromatographic quantificat ion of endogenous N-terminal Tyr-containing peptides in rat brain tiss ues. In this review article, liquid chromatographic procedures based o n the on-line postcolumn fluorogenic derivatization method with the th ree reagents are introduced with particular aims for the sensitive and reliable determinations of endogenous opioid oligopeptides and five f ragment peptides produced from precursor proteins in the tissues by tr yptic digestion in order to estimate the concentrations of opioid pept ide precursors, and further for the assays of endogenous enkephalin-ge nerating and degrading enzyme activities. (C) 1997 Elsevier Science B. V.