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 (