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| | Enzyme Inhibition Assay |
| Description: | Continuous assays were performed by recording the fluorescence increase at 405 nm (excitation@ 320 nm) induced by the cleavage of fluorogenic substrate, using 96-well nonbinding surface plates. Fluorescence signals were monitored using a Fluoroscan Ascent photon counter spectrophotometer (Thermo-Labsystems, Courtaboeuf, France) equipped with a temperature control device and a plate shaker. The substrate and enzyme concentrations for the experiments were chosen so as to remain well below 10% of substrate utilization and to observe initial rates. For each inhibitor, percentage inhibition was determined in triplicate experiments at five inhibitor concentrations, chosen to observe a 20-80% range of inhibition. Ki values were determined using the method proposed by Horovitz and Leviski, as described in Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 6654. |
| Affinity data for this assay | |
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