Assay Method Information

Assay Name:  Enzyme Inhibition Assay
Description:  The assays were performed in 384-well white opaque plates at 37 C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO4; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO4). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37 C.Test compounds were assayed over the range of concentrations from 10 μM to 20 μM. Test compounds were added to the enzymes and incubated for 30 minute at 37 C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37 C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).Plates were read on a fluorometer with excitation and emission wavelengths set to 320 nm and 405 nm, respectively. Inhibition constants were obtained by nonlinear regression of the data using the equation (GraphPad Software, Inc., San Diego, Calif.):v=v 0/[1+(I/K′)]where v is the reaction rate, v0 is the uninhibited reaction rate, I is the inhibitor concentration and K′ is the apparent inhibition constant.
Affinity data for this assay
 

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