| Assay Method Information | |
| | Human ACMSD-1 Inhibitor Assay |
| Description: | A solution of 7.8 μg/ml 3-hydroxyanthranilate 3,4-dioxygenase (3-HAO) with protein dilution buffer (50 mM 4-Morpholineethanesulfonic acid MES pH 6.0) and a solution of 6 μg/ml Human 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (human ACMSD) with protein dilution buffer (50 mM MES pH 6.0) were prepared separately. A serial 2-fold dilution of test compounds, from 512 nM until 0.5 nM were prepared. To a 96-well plate was added 50 μl of 7.8 μg/ml 3-HAO and 50 μl of 2× working solution (50 μM 3-hydroxyanthranilic acid (Sigma 148776), 2 mM ammonium iron(II) sulfate hexahydrate (Sigma V900031), in 50 mM MES pH 6.0) to start the reaction. The plate was placed into a SpectraMax Plus 384 Microplate Reader, Molecular Devices with the temperature set to 28° C. The absorbance at 360 nm for 10 min with 10 seconds interval was monitored and recorded. To a 96-well plate was added 50 μl of 6 μg/ml human ACMSD with series concentrations of the test compound. The absorbance was recorded at 360 nm for 10 min with 10 seconds interval. The data was analyzed in GraphPad Prism 6. A four-parameter dose-response curve was fitted. “Hill Slope” was constrained between −0.5 and −3, and such constrains was indicated when applied. |
| Affinity data for this assay | |
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