ABSTRACT
OBJECTIVE: To establish a highly effective HPLC method for analyzing 1-chlorine-2,4-dinitrobenzene (CDNB), and to determine the activity of GST enzyme in rat liver microsomes (RLM) by use of CDNB as probe and study its enzyme kinetics. METHODS: Welch Materials Ultimate™ XB C18 column (4.6 mm × 250 mm, 5 μm) was used, the mobile phase was acetonitrile-water(7:3), the flow rate was 0.8 mL · min-1, and the detection wavelength was 238 nm. CDNB was incubated with 0.02 mg · mL-1 protein in RLM at 37°C for 7 min and the reaction was terminated by cool acetonitrile. The reaction solution was centrifugated and the supernatant was filtered and analyzed by HPLC. The Vmax, Km and CLint were calculated by Sigma Plot. RESULTS: The retention time of CDNB was about 6 min without any interference. The lowest detection limit of CDNB was 1.0 μmol · L-1, and the calibration curve was linear from 2.5 to 100.0 μmol · L-1. The intra-day and inter-day relative standard deviations were less than 10% respectively. The method recoveries ranged from 99.38% to 108%. The reaction catalyzed by GST was terminated by acetonitrile and the incubation time was 7 min. The kinetic parameters, Vmax, Km, and CLint were 85.45 nmol · min-1 · (mg protein)-1, 15.09 μmol · L-1, and 5.73 mL · min-1 · (mg protein)-1, respectively. CONCLUSION: This method is reliable and the results can ac-curatelly indicate GST enzyme activity, which can be used for the kinetics study of GST in RLM.