A high pressure liquid chromatography-based assay for glutathione-S-transferase class distinction assay.
In order to expedite the process of classification of the members of the family of glutathione-S-transferases (GSTs) high performance liquid chromatography with photodiode array detection (HPLC-PDA) was used as a means for measuring enzymatic activity. The GST chosen for the development of the HPLC-PDA technique was from equine liver (E-GST). The characterizing substrates, ethacrynic acid (EA) and bromosulfophthalein (BSP), along with previously gathered characterization data allowed for the distinction of alpha, mu or pi-class enzymes. In an initial characterization of the previously unclassified E-GST it was determined that the enzyme was of the pi-class with specific activities of 0.062, +/-0.0015 micromol min(-1) mg(-1) and 0.0019, +/-0.00064 micromol min(-1) mg(-1) for EA and BSP, respectively. Finally, the activity of the E-GST with the EA and BSP substrates, was measured by HPLC-PDA, and was found to be 0.027, +/-0.003 micromol min(-1) mg(-1) and 0.002, +/-0.0005 micromol min(-1) mg(-1), respectively. While the HPLC-PDA data do not mirror the spectrophotometric results quantitatively the overall response by the E-GST was the same. In general, the E-GSTs were shown to belong to the pi-class when characterized by HPLC-PDA due to an EA specific activity greater than 0.01 micromol min(-1) mg(-1) and a negligible BSP activity (</=0.002 micromol min(-1) mg(-1)).
Blanchette
Brian N
BN
Department of Chemistry and Biochemistry, School of Marine Science and Technology, University of Massachusetts Dartmouth, 285 Old Westport Road, Dartmouth, MA 02747, United States.
Singh
Bal Ram
BR
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005982:Glutathione Transferase / Q000032:analysis* / Q000145:classification* / Q000378:metabolism; D006736:Horses; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D013053:Spectrophotometry; D013379:Substrate Specificity
D005982:Glutathione Transferase