Analysis of glutathione S-transferase-catalyzed S-alkylglutathione formation by high-performance liquid chromatography

✍ Scribed by James W. Tracy; Kathleen A. O'Leary

• Book ID: 102986387
• Publisher: Elsevier Science
• Year: 1991
• Tongue: English
• Weight: 572 KB
• Volume: 193
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(91)90034-q

✦ Synopsis

Metabolism of alkyl halides and some organophosphorous compounds by glutathione S-transferases (EC 2.5.1.18) leads to formation of an S-alkylglutathione as a common product. We have developed an HPLC assay for formation of S-methylglutathione and S-ethylglutathione that is applicable to measuring enzyme activity toward a variety of xenobiotic substrates. The conjugates are derivatized with 1-fluoro-2,4-dinitrobenzene to form the corresponding N-2,4-dinitrophenyl derivatives, which are then separated by reverse-phase HPLC with gradient elution. The utility of the method is illustrated by the use of partially purified preparations of rat liver glutathione S-transferases and several prototypic substrates including iodomethane, iodoethane, dichlorvos, and methyl parathion. The limit of detection is about 50 pmol of N-(2,4-dinitrophenyl)-S-alkylglutathione. Advantages of the method over other assays of S-alkyl transferase activity are discussed.