An online capillary electrophoresis (CE)-based thrombin (THR) immobilized enzyme microreactor (IMER) method was established to screen THR inhibitors in this study. S-2366 was used as chromogenic substrate for determination of THR activity and other kinetic constants. After continuously run for 50 times, the prepared IMER could still remain 89% of the initial immobilized enzyme activity. The Michaelis-Menten constant (K_m) of immobilized THR was measured as 0.514 mmol/L and the half-maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) of argatroban on THR were determined as 78.07 and 26.53 nmol/L, respectively, which indicated that CE-based THR IMER was successfully established and could be applied to screen THR inhibitors. Then the prepared IMER was used to investigate the inhibitory potency on THR of four main catechins in green tea including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). The results showed that ECG and EGCG had good THR inhibition activity and their inhibition rates at concentration of 200 μmol/L were 53.2 ± 3.8% and 55.8 ± 2.6%, respectively, which was in consistent with the results of microplate reader assay. Additionally, molecular docking results showed that the benzopyran groups of ECG and EGCG were inserted into the THR active pocket and interacted with residues LYS60F, TRP60D, TRY60A, IEU99, GLY216, HIS57 and SER195, but EC and EGC did not. Therefore, the developed CE-based THR IMER is reliable method for measuring THR inhibitory activity of natural inhibitors.

建立了基于在线毛细管电泳（CE）的凝血酶（THR）固定化酶微反应器（IMER）的方法，以筛选本研究中的THR抑制剂。S-2366被用作生色底物，用于测定THR活性和其他动力学常数。连续运行50次后，制得的IMER仍可保持初始固定酶活性的89％。固定的THR的Michaelis-Menten常数（K _m）为0.514 mmol / L，半数最大抑制浓度（IC ₅₀）和抑制常数（K _i阿加曲班对THR的测定值分别为78.07和26.53 nmol / L，表明成功建立了基于CE的THR IMER，可用于筛选THR抑制剂。然后使用制得的IMER来研究绿茶中的四种主要儿茶素（包括表儿茶素（EC），表没食子儿茶素（EGC），表儿茶素没食子酸酯（ECG）和表没食子儿茶素没食子酸酯（EGCG）对THR的抑制力。结果表明，ECG和EGCG具有良好的THR抑制活性，在200μmol/ L的浓度下其抑制率分别为53.2±3.8％和55.8±2.6％，与酶标仪检测结果相符。此外，分子对接结果表明，ECG和EGCG的苯并吡喃基团已插入THR活性囊中，并与残基LYS60F，TRP60D，TRY60A，IEU99，GLY216，HIS57和SER195，但EC和EGC没有。因此，开发的基于CE的THR IMER是测量天然抑制剂THR抑制活性的可靠方法。