Studies on glutathione-S-arene oxidase transferase—A sensitive assay and partial purification of the enzyme from sheep liver

A sensitive assay has been devised for glutathione- S -arene oxidase transferase using as substrates naphthalene-1,2-oxide or styrene oxide along with [ 35 S]glutathione. Activity of the order of 2–3 nmoles of conjugate formed during a 5-min incubation can be detected. This yields about 2000 cpm above a blank of about 1500 cpm. Transferase activity was found mainly in liver and kidney but was also present in most other tissues of rats. Glutathione- S -arene oxide transferase has been purified 70- to 80-fold from sheep liver 100,000 g supernatants using the conventional procedures. After electrofocusing, enzyme activity separated into two major peaks and two or three minor peaks, ranging in isoelectric point from pH 6.5 to 7.5. Activities assayed with naphthalene-1,2-oxide or styrene oxide as substrates were found to almost parallel each other in all the peaks. The sheep liver transferase required neither metal ions nor cofactors such as FAD, pyridoxal-phosphate and thiamine pyrophosphate. The molecular weight of the transferase has been estimated to be about 40,000. K m values for glutathione, naphthalene-1,2-oxide, and styrene oxide are 1.6, 0.11, and 0.13 m m , respectively. K m values for glutathione decreased with increasing pH, whereas the K m values for naphthalene-1,2-oxide were independent of pH in the range of 6.5–8.