Purification, characterization and the effect of some drugs on glutathione s-transferase (GST) enzyme from rat erythrocyte

In this study, glutathione S-transferase (GST; EC: 2.5.1.18) enzyme was purified from rat erythrocyte in a single step by glutathione agarose affinity chromatography. The enzyme was purified with 6.3 EU/mg protein specific activity, with 44% recovery and 115 purification fold. The purity of enzyme was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and produced a single protein band with a calculated molecular weight of 25.2 kDa. The characterization studies showed that the optimum pH=8 in potasium phosphate buffer, optimum temperature is 50oC, optimum ionic strength is 0.2M in potasium phosphate and stable pH=8 in potasium phosphate buffer. Kinetic studies performed for calculating KM and Vmax for (GST) using both glutathione (GSH), and 1-chloro 2,4- dinitrobenzene (CDNB) as two subsrates, the obtained results are 1.22 mM and 1.21 EU/mL respectively for GSH 0.374 mM and 3.614 EU/mL respectively for (CDNB). Finally inhibition or activation effect of some drugs on the enzyme activity were studied in vitro. The results proved that buscopan and ampisid were two activators of the enzyme, gentamicin and clindamicin were two inhibitors (IC50 values were found as 1.69 and 6.9 mM respectively and Ki values were found as 1.70 and 2.36 mM respectively), and cefazoline has no effect on the enzyme activity.