title: Capillary electrophoresis applied to screening of trypsin inhibitors using microreactor with trypsin immobilized by glutaraldehyde.
abstract: An efficient and durable online capillary immobilized trypsin microreactor was successfully established to study the enzyme kinetics of trypsin and screen its inhibitors from natural extracts through capillary electrophoresis (CE). In this procedure, trypsin was immobilized on the inner wall at the inlet of the capillary treated with 3-aminopropyltrimethoxy silane (3-APTES), producing a trypsin microreactor via cross-linking of glutaraldehyde with 3-APTES and trypsin. The rest of the capillary was selected as a channel for separating the generated product and unreacted substrate of the trypsin enzymatic reaction. The parameters affecting the separation efficiency and activity of immobilized trypsin were evaluated systematically. The optimized conditions were as follows: 50 mM Tris-HCl (pH 8.0), 15 kV, 37 °C, 10 mM substrate, incubation for 2 min. Under optimal conditions, separation of the product and substrate was achieved through CE within 3.5 min. The obtained results of Michaelis constant, inhibition kinetics constant, and half-maximal inhibitory concentration for the immobilized trypsin using benzamidine hydrochloride hydrate as a model inhibitor were 1.56, 1.79 and 3.98 mM, respectively. The proposed method was successfully applied for screening of trypsin inhibitors from 19 kinds of natural extracts.
authors.affiliation: College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China.
authors.forename: Wenao
authors.initials: W
authors.lastname: Min
authors.forename: Shumin
authors.initials: S
authors.lastname: Cui
authors.forename: Weiping
authors.initials: W
authors.lastname: Wang
authors.forename: Jianrong
authors.initials: J
authors.lastname: Chen
authors.forename: Zhide
authors.initials: Z
authors.lastname: Hu
mesh_terms: D000818:Animals; D001688:Biological Products / Q000302:isolation & purification / Q000494:pharmacology; D002417:Cattle; D004353:Drug Evaluation, Preclinical; D004553:Electric Conductivity; D004573:Electrolytes; D019075:Electrophoresis, Capillary / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D005976:Glutaral / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D055616:Microtechnology / Q000379:methods*; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D001688:Biological Products; D004573:Electrolytes; D004800:Enzymes, Immobilized; D010936:Plant Extracts; D014361:Trypsin Inhibitors; D014357:Trypsin; D005976:Glutaral