CHROMATOGRAPHIC-SEPARATION OF FLUORESCENT THIOL ADDUCTS OF 4-CHLORO-7-SULPHOBENZOFURAZAN - USE AS SUBSTRATES FOR ENZYMES OF THE MERCAPTURICACID XENOBIOTIC PATHWAY

Fluorescent adducts of 4-chloro-7-sulphobenzofurazan with cysteine, cy steinylglycine, reduced glutathione and N-acetylcysteine were prepared . Adducts were separated by HPLC on a 3-mm Nova-Pak C-18 reversed-phas e column using isocratic elution with a solvent of acetonitrile-0.15 M phosphoric acid (5:95) buffered at pH 2.5. The adducts were detected using a fluorescence detector set at an excitation wavelength of 365 n m and an emission wavelength of 510 nm and an ultraviolet detector at 254 nm. The adduct of reduced glutathione was also formed by the actio n of the enzyme glutathione-S-transferase. This adduct acted as a subs trate for the enzyme gamma-glutamyltranspeptidase and the product of t his reaction, the 4-chloro-7-sulphobenzofurazanyl derivative of cystei nylglycine, acted as a substrate for either dipeptidase or aminopeptid ase M. The sequential enzymic effects could be detected by changes in the relative fluorescence intensity of the solutions to which the resp ective enzymes had been added but were more appropriately followed by changes in the HPLC elution profiles after enzymic treatment of soluti ons. (C) 1998 Elsevier Science B.V.