title: Automated method, based on micro-sequential injection, for the study of enzyme kinetics and inhibition.
abstract: A micro-reactor system with continual spectrophotometric detection has been operated in Sequential Injection lab-on-valve (SI-LOV) mode and applied to enzyme kinetics and inhibition studies, using acetylcholinesterase (AChE) and angiotensin-converting enzyme (ACE) as model systems. With the advantages of automation, real-time kinetic measurement, and thorough mixing, the SI-LOV micro-reactor system allows for the monitoring of initial reaction rates and determination of reactant concentrations in the reaction mixture, both of which are essential for the determination of kinetic constants for enzymes and inhibitors. Enzyme, substrate, and inhibitor are precisely metered by the syringe pump and delivered to a stirred micro-reactor, followed by a reference scan that establishes the baseline for the following reaction rate measurement. Michaelis constants (Km) for AChE and ACE were determined to be 0.16 mM and 0.30 mM, respectively, which are consistent with literature values. The type of inhibition (competitive, uncompetitive, or mixed), the dissociation constants for the inhibitors, and the inhibitor dose-response curves were also determined.
authors.affiliation: Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195, USA. kate_2002_m@yahoo.com
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Carroll
authors.forename: Louis
authors.initials: L
authors.lastname: Scampavia
authors.forename: Jaromir
authors.initials: J
authors.lastname: Ruzicka
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000187:drug effects*; D001322:Autoanalysis; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004491:Edrophonium / Q000494:pharmacology; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods; D007700:Kinetics; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000187:drug effects*; D012680:Sensitivity and Specificity; D013594:Syringes; D013619:Tacrine / Q000494:pharmacology; D013997:Time Factors
chemical_list: D004151:Dipeptides; C038123:alanylproline; D013619:Tacrine; D004491:Edrophonium; D000110:Acetylcholinesterase; D007703:Peptidyl-Dipeptidase A