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Assay Method Information

Assay Name:  In Vitro Enzyme Inhibition Assay
Description:  The ability of the compounds disclosed herein to inhibit human plasma kallikrein activity was quantified according to the procedures below.A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4 test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4hPK and 2PFR-AMC, respectively.60 μL of each 4 test compound buffer stock was combined with 60 μL of 4hPK to yield 120 μL of 2 test compound buffer stock/2hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37 C. To each well, 50 μL of pre-warmed 2PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37 C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50.
Affinity data for this assay
 

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Last update November 1, 2007
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