Assay Method Information

Assay Name:  Inhibitory Activity
Description:  The pharmacological activity of compounds disclosed herein was tested in the following screen (Test A) in which the compounds were tested in the presence of ascorbate, which reacts with MPO-derived hypochlorous acid (HOCl) to form dehydro-ascorbate. The loss of ascorbate is followed by measuring absorbance at 260 nm.Assay buffer: 100 μM diethyl triamine pentaacetic acid (DTPA) in buffer consisting of 10 mM Na2HPO4/NaH2PO4, 3 mM KCl in 140 mM NaCl, pH 7.4.Enzyme solution: MPO purified from the human cell line HL60, 1.38 nM (final concentration 0.7 nM) and L-ascorbate, 100 μM (final concentration 50 μM) in Assay bufferSubstrate solution: H2O2, 98 μM (final concentration 49 μM) Forty μL of the enzyme solution was added to 0.6 μL compound serially diluted in DMSO. Absorbance was measured at 260 nm to obtain a compound blank value. After an additional 10 min, 40 μL of the substrate solution was added and the absorbance at 260 nm was recorded between 4 and 40 min to obtain kinetic readings of enzyme activity.
Affinity data for this assay
 

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