Analysis of glutathione S-transferase-catalyzed S-alkylglutathione formation by high-performance liquid chromatography.
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.
Tracy
J W
JW
Department of Comparative Biosciences, University of Wisconsin-Madison 53706.
O'Leary
K A
KA
D019297:2,4-Dinitrophenol; D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004140:Dinitrophenols; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D051381:Rats; D013379:Substrate Specificity
D004140:Dinitrophenols; C042431:S-ethyl glutathione; D005982:Glutathione Transferase; D005978:Glutathione; C017514:S-methyl glutathione; D019297:2,4-Dinitrophenol