| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | The compounds were tested for enzyme inhibition using fluorescence resonance energy transfer (FRET) assay. Fluorescence measurements were performed in a CytoFluor multiwell plate reader, series 4000. Cleavage of internal quenched substrate liberates emission-active Mca product, causing an increase in fluorescence emission at 395 nM (the excitation wavelength is 330 nM). The rate of emission change is proportional to enzyme activity. Initial velocities, in the presence or absence of inhibitor, were measured as slopes of the linear portion of the product progress curves. Apparent Ki values were determined by plotting the inhibitor concentration dependence of the fractional velocity for each enzyme, and fitting the data by a tight binding inhibition equation. |
| Affinity data for this assay | |
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