| Assay Method Information | |
| | HPLC Assay |
| Description: | For selected lead compounds from fluorescence-based high-throughput screening, Km and Vmax were calculated using the double-reciprocal Lineweaver-Burk plot of rate versus substrate concentration, the values of Ki was obtained using replots of inhibitor concentration against Km/Vmax and I/Vmax. To eliminate false positives resulting from fluorescence quenching by test compounds, high-performance liquid chromatography(HPLC) was used to separate the cleavage product and inhibitor compounds. The IC50 values were evaluated using six different concentrations of the inhibitor ranging from 0.3 to 40 ug/mL and measuring the fluorescence peak of the C-terminal cleavage product. |
| Affinity data for this assay | |
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