HPLC测定大鼠肝微粒体中谷胱甘肽硫转移酶活性及体外动力学研究

 目的 建立一种测定1-氯-2，4-二硝基苯（CDNB）高效液相色谱分析方法，并以1-氯-2，4-二硝基苯为探针测定大鼠肝微粒体中谷胱甘肽硫转移酶（GST）活性并进行体外动力学分析。方法 色谱条件：Welch Materials Ultimate^TM XB C₁₈反相柱（4.6 mm×250 mm，5 μm），流动相：乙腈-水（7∶3)，流速0.8 mL·min^-1，柱温30 ℃，检测波长238 nm。实验方法：1-氯-2，4-二硝基苯与大鼠肝微粒体在37 ℃温孵7 min后，冰乙腈终止反应。反应液离心取上清液过滤后进行HPLC分析，通过Sigma Plot 软件作图求算V_max、K_m及代谢清除率（CL_int）值。结果 1-氯-2，4-二硝基苯的R_t=6.0 min，峰形良好，且无内源性干扰。最低检测限为1.0 μmol·L^-1，线性范围：2.5～100.0 μmol·L^-1。日内、日间精密度均小于10%。5 d内于室温及-20 ℃下较稳定。方法回收率为99.38%～108%。动力学分析表明，不同浓度1-氯-2，4-二硝基苯在0.02 mg·mL^-1蛋白浓度下孵育7 min，测得动力学参数：V_max为85.45 nmol·min^-1·（mg protein）^-1；K_m为15.09 μmol·L^-1； CL_lint为5.66 mL·min^-1·（mg protein）^-1。结论 该方法稳定可靠，灵敏度高，能准确快速测定谷胱甘肽硫转移酶活性，可用于其体外动力学研究。

Determination of GST Activity in Rat Liver Microsomes Using HPLC and Study on Its Kinetics in Vitro

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 TM 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 ℃ 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 accuratelly indicate GST enzyme activity,which can be used for the kinetics study of GST in RLM.