High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate β-lyase activity: Application to S-1,2-dichlorovinyl- l-cysteine and S-2-benzothiazolyl- l-cysteine

An HPLC—fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate β-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid, specific, and very sensitive. The assay has been evaluated with two model substrates for rat renal cytosolic β-lyase, notably S-1,2-dichorovinyl- l-cysteine (DCVC) and S-2-benzothiazolyl- l-cysteine (BTC). Equimolar formation of pyruvic acid and 2-mercaptobenzothiazole, a chromophoric thiol, indicated that pyruvic acid formation actually reflects the β-elimination activity of β-lyase during the β-elimination of BTC. From this it follows that the pyruvic acid assay can be applied to the measurement of the β-elimination activity of this enzyme, independent of the presence of chromophoric groups or radiolabels in substrates. Due to the large linear range and the very high sensitivity of the present HPLC-fluorescence assay (detection limit, 7.5 pmol of pyruvic acid), both good and poor substrates of β-lyase can be measured. Enzyme kinetic data are presented for the model substrates BTC and DCVC and for four structurally related S-2,2-difluoroethyl- l-cysteine conjugates.