Analytical Sciences
Online ISSN : 1348-2246
Print ISSN : 0910-6340
ISSN-L : 0910-6340
Original Papers
Automated Method, Based on Micro-Sequential Injection, for the Study of Enzyme Kinetics and Inhibition
Yan CHENAndrea D. CARROLLLouis SCAMPAVIAJaromir RUZICKA
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JOURNAL FREE ACCESS

2006 Volume 22 Issue 1 Pages 9-14

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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.
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© 2006 by The Japan Society for Analytical Chemistry
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