Chromatographic separation of fluorescent thiol adducts of 4-chloro-7-sulphobenzofurazan. Use as substrates for enzymes of the mercapturic acid xenobiotic pathway.
Fluorescent adducts of 4-chloro-7-sulphobenzofurazan with cysteine, cysteinylglycine, reduced glutathione and N-acetylcysteine were prepared. Adducts were separated by HPLC on a 3-mm Nova-Pak C18 reversed-phase 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 nm and an emission wavelength of 510 nm and an ultraviolet detector at 254 nm. The adduct of reduced glutathione was also formed by the action of the enzyme glutathione-S-transferase. This adduct acted as a substrate for the enzyme gamma-glutamyltranspeptidase and the product of this reaction, the 4-chloro-7-sulphobenzofurazanyl derivative of cysteinylglycine, acted as a substrate for either dipeptidase or aminopeptidase M. The sequential enzymic effects could be detected by changes in the relative fluorescence intensity of the solutions to which the respective enzymes had been added but were more appropriately followed by changes in the HPLC elution profiles after enzymic treatment of solutions.
Chen
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XP
School of Chemical Sciences, Swinburne University of Technology, Hawthorn, Victoria, Australia.
Cross
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Clark
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AG
Baker
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WL
D000111:Acetylcysteine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001583:Benzoxazoles / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D004798:Enzymes / Q000378:metabolism*; D005453:Fluorescence; D005978:Glutathione / Q000737:chemistry; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000302:isolation & purification*; D015262:Xenobiotics / Q000378:metabolism*
D001583:Benzoxazoles; D004151:Dipeptides; D004798:Enzymes; D013438:Sulfhydryl Compounds; D015262:Xenobiotics; C028505:cysteinylglycine; C034293:4-chloro-7-sulfobenzofurazan; D005978:Glutathione; D003545:Cysteine; D000111:Acetylcysteine