Evaluation of thrombin inhibitory activity of catechins by online capillary electrophoresis-based immobilized enzyme microreactor and molecular docking.

Autor: Li QQ; School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, PR China., Yang FQ; School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, PR China. Electronic address: fengqingyang@cqu.edu.cn., Wang YZ; School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, PR China., Wu ZY; School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, PR China., Xia ZN; School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, PR China., Chen H; School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, PR China.
Jazyk: angličtina
Zdroj: Talanta [Talanta] 2018 Aug 01; Vol. 185, pp. 16-22. Date of Electronic Publication: 2018 Mar 18.
DOI: 10.1016/j.talanta.2018.03.049
Abstrakt: 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 (IC 50 ) 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.
(Copyright © 2018 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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