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 and body fluids. Their endogenous concentrations are controlled by synthetic and metabolic enzymes. It is therefore of great importance to develop an analytical protocol that permits facile and sensitive determination of various opioid peptides and related enzyme activities. Several chemical reactions with fluorogenic reagents have been developed for liquid Chromatographic detection of peptides, increasing either sensitivity or selectivity. Among them, a fluorogenic chemical reaction using three reagents such as hydroxylamine, cobalt(II) and borate salts which provides fluorescent derivatives for only N-terminal Tyr-containing peptides is the most applicable to the Chromatographic quantification of endogenous N-terminal Tyr-containing peptides in rat brain tissues. In this review article, liquid Chromatographic procedures based on the on-line postcolumn fluorogenic derivatization method with the three reagents are introduced with particular aims for the sensitive and reliable determinations of endogenous opioid oligopeptides and five fragment peptides produced from precursor proteins in the tissues by tryptic digestion in order to estimate the concentrations of opioid peptide precursors, and further for the assays of endogenous enkephalin-generating and degrading enzyme activities.