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Assay Method Information

Assay Name:  Enzyme Inhibition Assay
Description:  Potential inhibitors were evaluated using the progress curve method. Assays were carried out in the presence of variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of inhibitor and substrate. Product fluorescence (excitation at 360 nM, emission at 460 nM) was monitored with a Perceptive Biosystems Cytofluor II fluorescent plate reader. Product progress curves were generated over 20-30 min following formation of AMC product. For those compounds whose progress curves were linear, apparent inhibition constants (Ki,app) were calculated according to the equation: V = VmA[Ka(1 + I/Ki,app) + A], where V is the velocity of the reaction with maximal velocity Vm, A is the concentration of substrate with Michaelis constant of Ka, and I is the concentration of inhibitor.
Affinity data for this assay
 

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Last update November 1, 2007
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