title: Monobromobimane as an affinity label of the xenobiotic binding site of rat glutathione S-transferase 3-3.
abstract: Monobromobimane (mBBr), besides being a substrate in the presence of glutathione, inactivates rat liver glutathione S-transferase 3-3 at pH 7.5 and 25 degrees C as assayed using 1-chloro-2,4-dinitrobenzene (CDNB). The rate of inactivation is enhanced about 5-fold by S-methylglutathione. Substrate analogs bromosulfophthalein and 2,4-dinitrophenol decrease 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 added protectant 2,4-dinitrophenol yields an enzyme that is catalytically active and contains only 0.89 mol of reagent/mol of subunit. mBBR-modified enzyme is fluorescent, and fluorescence energy transfer occurs between intrinsic tryptophan and covalently bound bimane in modified enzyme. Both Tyr115 and Cys114 are modified, but Tyr115 is the initial reaction target and its modification correlates with loss of activity toward CDNB. The fact that the activity toward mBBr is retained by the enzyme after modification suggests that rat isozyme 3-3 has two binding sites for mBBr.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
authors.forename: L
authors.initials: L
authors.lastname: Hu
authors.forename: R F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000378:metabolism; D015262:Xenobiotics / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001643:Bridged Bicyclo Compounds; D007527:Isoenzymes; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D015262:Xenobiotics; D005982:Glutathione Transferase; C028381:monobromobimane