Ammonium 4-chloro-7-sulfobenzofurazan: a fluorescent substrate highly specific for rat glutathione S-transferase subunit 3.
Ammonium 4-chloro-7-sulfobenzofurazan (Sbf-Cl) is a water-soluble fluorescent reagent which is highly specific for thiol groups. We describe here a simple and sensitive fluorescence assay which is highly specific for subunit 3 of the rat glutathione S-transferases. Specific activities of isoenzymes 3-3 and 3-4 were an order of magnitude greater than those of isoenzymes 1-1, 1-2, 2-2, and 4-4, with glutathione and Sbf-Cl concentrations of 1 and 2 mM, respectively. The catalytic specificity constant, kcat/Km, was in the range of 10(4)-10(6) M-1 s-1, indicating that Sbf-Cl is a very good substrate for rat hepatic glutathione S-transferases. The specificity constants measured for the heterodimers 1-2 and 3-4 were greater than the values predicted when dimeric independence is assumed. This suggests that binding of Sbf-Cl to the glutathione S-transferases may result in steric alterations and subsequent elevation in enzymatic activity. Sbf-Cl was shown to be at least 10-fold more sensitive for the detection of rat GST isoenzyme 3-3 than DCNB. Consequently, Sbf-Cl will have an important future role in the investigation of the active site topology of glutathione S-transferases, in addition to its obvious potential as a substrate for the detection and quantitation of rat glutathione S-transferase subunit 3.
Bolton
R M
RM
Key Centre for Applied and Nutritional Toxicology, RMIT-University, Melbourne, Victoria, Australia.
Haritos
V S
VS
Whitehouse
M W
MW
Ahokas
J T
JT
D000818:Animals; D001583:Benzoxazoles / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D009578:Nitrobenzenes / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D007527:Isoenzymes; D046911:Macromolecular Substances; D009578:Nitrobenzenes; C034293:4-chloro-7-sulfobenzofurazan; C053747:2,4-dichloro-1-nitrobenzene; D005982:Glutathione Transferase; D005978:Glutathione