Abstract
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 (Km) of immobilized THR was measured as 0.514?mmol/L and the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) 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.
… 2. Materials and methods. 2.1. Reagents. Bovine THR was obtained from Sigma-Aldrich(Shanghai) Trading Co., Ltd. (Shanghai, China).
EC,
EGC,
ECG, and
EGCG were purchased from Chengdu
Biopurify Phytochemicals Ltd. (Chengdu, China) …
