title: High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme.
abstract: A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.
authors.affiliation: Faculty of Chemistry, Northeast Normal University, 5368 Renmin Street, Changchun, Jilin 130024, PR China. yangl330@nenu.edu.cn
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Chunyang
authors.initials: C
authors.lastname: Chen
authors.forename: Yuanfang
authors.initials: Y
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Shi
authors.forename: Sidong
authors.initials: S
authors.lastname: Liu
authors.forename: Liping
authors.initials: L
authors.lastname: Guo
authors.forename: Haifeng
authors.initials: H
authors.lastname: Xu
mesh_terms: D000809:Angiotensins / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D057075:Enzyme Assays / Q000295:instrumentation* / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D007834:Lasers; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000809:Angiotensins; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D014357:Trypsin