MONOBROMOBIMANE AS AN AFFINITY LABEL OF THE XENOBIOTIC BINDING-SITE OF RAT GLUTATHIONE-S-TRANSFERASE-3-3

Monobromobimane (mBBr), besides being a substrate in the presence of g lutathione, inactivates rat liver glutathione S-transferase 3-3 at pH 7.5 and 25 degrees C as assayed using 1-chloro-2,4-dinitrobenzene (CDN B). The rate of inactivation is enhanced about 5-fold by S-methylgluta thione. Substrate analogs bromosulfophthalein and 2,4-dinitrophenol de crease the rate of inactivation at least 20-fold. Upon incubation for 60 min with 0.25 mM mBBr and S-methylglutathione, the enzyme loses 91% of its activity toward CDNB and incorporates 2.14 mol of reagent/mol of subunit, whereas incubation under the same conditions but with adde d protectant 2,4-dinitrophenol yields an enzyme that is catalytically active and contains only 0.89 mol of reagent/mol of subunit. mBBr-modi fied enzyme is fluorescent, and fluorescence energy transfer occurs be tween intrinsic tryptophan and covalently bound bimane in modified enz yme. Both Tyr(115) and Cys(114) are modified, but Tyr(115) is the init ial reaction target and its modification correlates with loss of activ ity toward CDNB. The fact that the activity toward mBBr is retained by the enzyme after modification suggests that rat isozyme 3-3 has two b inding sites for mBBr.