Abstract

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"High-throughput enzyme kinetics: Simultaneous determination of tight-binding inhibition constants and enzyme concentration" Kuzmic, P.; Elrod, K.C.; Cregar, L.M.; Sideris, S; Rai, R.; and Janc, J.W. (2000) Anal. Biochem. 286, 45-50. Abstract: Active site titration by a reversible tight-binding inhibitor normally depends on prior knowledge of the inhibition constant. Conversely, the determination of tight-binding inhibition constants normally requires prior knowledge of the active enzyme concentration. Often, neither of these quantities is known with sufficient accuracy. This paper describes experimental conditions under which both the enzyme active site concentration and the tight-binding inhibition constant can be determined simultaneously from a single dose-response curve. A suitable computer program for performing the analysis is available electronically at www.biokin.com . Representative experimental data are shown for the inhibition of human kallikrein.

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