7-Glycidoxycoumarin (GOC): a fluorophotometric epoxide substrate for the assay of glutathione S-transferase activity.
7- Glycidoxycoumarin ( GOC ), a new fluorophotometric epoxide substrate for glutathione S-transferase (GSH TFase ), was conjugated regiospecifically with GSH at pH 6.5 in rat liver cytosol to yield S-(2-hydroxy-3-(7'- coumaroxy )-1-propyl)glutathione which was isolated by HPLC and identified with an authentic specimen by 13C NMR spectroscopy. The conjugation product formed in the incubation media consisting of GOC , GSH, and 9000 g supernatant fractions from various tissues of the rat, was directly determined by photometry of fluorescence emission at 388 nm at an excitation wavelength of 328 nm after removal of the unreacted substrate and its enzymic hydrolysis product, 7-(1',2'-dihydroxy-3'-propoxy)coumarin, by simple extraction with isobutyl alcohol in the presence of a saturating amount of sodium chloride. Stability of GOC at pH 6.5 markedly retarded its autoconjugation with GSH and made the fluorophotometric method sensitive enough to assay small GSH TFase activities in gel column chromatographic fractions as well as in various tissues of the animals. Apparent Km and Vmax for GOC in rat liver cytosol were 55 microM and 7.41 nmole/mg protein/min, respectively. GSH conjugation of GOC was catalyzed by at least two isozymes, E and AA, of hepatic GSH TFases .
D000818:Animals; D003374:Coumarins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000032:analysis*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence / Q000379:methods*
D003374:Coumarins; C041461:7-glycidoxycoumarin; D005982:Glutathione Transferase; D005978:Glutathione