| Assay Method Information | |
| | Kallikrein Inhibition Assay Utilizing a Quenched AMC Peptide Substrate |
| Description: | A fluorescence intensity (FLINT) based assay was used to monitor inhibition of human plasma kallikrein. The peptide substrate, Z-Gly-Pro-Arg-AMC (Purchased from Bachem; Catalog #I-1150) was chosen based on its relatively low KM for kallikrein which enables running the assay at lower substrate concentrations to control background fluorescence. The kinetic parameters for this substrate were determined by fitting titration data to the Michaelis-Menten equation yielding a KM=40 μM, kcat=0.76 s−1, and kcat/KM=18932 M−1s−1. The Kallikrein FLINT assay was used with the following Z-Gly-Pro-Arg-AMC assay buffer: 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM CaCl2), 0.1 mg/mL BSA, 0.03% CHAPS. Assay buffer was prepared by mixing all ingredients fresh. 2×Z-Gly-Pro-Arg-AMC peptide substrate was prepared by diluting 10 mM stock into 1×assay buffer for a 100 μM working concentration (50 μM final concentration). The 2×kallikrein stock solution was prepared by diluting 14.76 μM stock in 1×assay buffer for a 4 nM working stock solution (2 nM final concentration). |
| Affinity data for this assay | |
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