High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate beta-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 beta-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 beta-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 beta-elimination activity of beta-lyase during the beta-elimination of BTC. From this it follows that the pyruvic acid assay can be applied to the measurement of the beta-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 beta-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.
Stijntjes
G J
GJ
Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, The Netherlands.
te Koppele
J M
JM
Vermeulen
N P
NP
D000818:Animals; D052160:Benzothiazoles; D013437:Carbon-Sulfur Lyases; D002458:Cell Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis; D003600:Cytosol / Q000201:enzymology; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008190:Lyases / Q000032:analysis / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D011773:Pyruvates / Q000032:analysis*; D019289:Pyruvic Acid; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods
D052160:Benzothiazoles; D011773:Pyruvates; C034680:S-(2-benzothiazolyl)cysteine; C039961:S-(1,2-dichlorovinyl)cysteine; D019289:Pyruvic Acid; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C019969:S-alkylcysteine lyase; D003545:Cysteine