To identify the inhibitor of glutathione S-transferase（GST）,a high-throughput screening method was established in a 384-well microplate with total 35 μL volume,and the absorbance at 340 nm is detected.The concentra...To identify the inhibitor of glutathione S-transferase（GST）,a high-throughput screening method was established in a 384-well microplate with total 35 μL volume,and the absorbance at 340 nm is detected.The concentrations of substrates,CDNB and GST were determined by chromatometry.The optimal enzyme kinetics reaction time and temperature are 2 h and 30 ℃,respectively.The established model was evaluated by NaOCl,a known GST inhibitor,and the parameter Z′ was 0.77,which showed a high feasibility and stability of the assay.A total of 31 098 compounds were screened,of which 4 compounds were shown to inhibit GST activity,high inhibiting activity for their IC50 of GST inhibition was 3.94,4.05,74.85,and 77.41 mg·L-1,separately.The results indicated that the colorimetric method by using CDNB and GSH as substrate is stable,sensitive,reproducible and also suitable for high throughput screening.展开更多
文摘To identify the inhibitor of glutathione S-transferase（GST）,a high-throughput screening method was established in a 384-well microplate with total 35 μL volume,and the absorbance at 340 nm is detected.The concentrations of substrates,CDNB and GST were determined by chromatometry.The optimal enzyme kinetics reaction time and temperature are 2 h and 30 ℃,respectively.The established model was evaluated by NaOCl,a known GST inhibitor,and the parameter Z′ was 0.77,which showed a high feasibility and stability of the assay.A total of 31 098 compounds were screened,of which 4 compounds were shown to inhibit GST activity,high inhibiting activity for their IC50 of GST inhibition was 3.94,4.05,74.85,and 77.41 mg·L-1,separately.The results indicated that the colorimetric method by using CDNB and GSH as substrate is stable,sensitive,reproducible and also suitable for high throughput screening.
