Ammonium 4-Chloro-7-sulfobenzofurazan: A Fluorescent Substrate Highly Specific for Rat Glutathione S-Transferase Subunit 3
✍ Scribed by R.M. Bolton; V.S. Haritos; M.W. Whitehouse; J.T. Ahokas
- Book ID
- 102562867
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 468 KB
- Volume
- 216
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 \mathrm{~mm}), respectively. The catalytic specificity constant, (k_{\text {cat }} / K_{m}), was in the range of (10^{4}-10^{6} \mathrm{~m}^{-1}) (\mathbf{s}^{-1}), indicating that (\mathrm{Sbf}-\mathrm{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. c) 1994 Academic Press, Inc.