Assay Method Information

Assay Name:  Enzyme Inhibition Assay
Description:  Kinetic inhibition of human thrombin was determined photometrically at 405 nm using the chromogenic substrate Pefachrom tPa. Reactions were performed under the conditions using different concentrations of substrate (182, 91 and 45 uM) and inhibitor (36.4, 27.3, 18.2 and 9.1 uM for the weakest inhibitor and 3.6, 2.7, 1.8 and 0.9 nM for the tightest binder). Activity of thrombin was adjusted by diluting solution with 154 mM NaCl until linear conversion of the substrate could be detected over 5 min in an appropriate absorption window (~0.2-0.8). The assay was stopped after 3 min with concentrated acetic acid and absorption in each well was corrected for the blank value. Ki values were determined as described by Dixon.
Affinity data for this assay

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