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Assay Method Information

Assay Name:  APAH Fluorimetric Assay
Description:  Activity assays were conducted at 25 C and contained 250 nM APAH (∼50% Zn2+ occupancy), 150 μM substrate, and 0−250 μM inhibitor in assay buffer [25 mM Tris (pH 8.2), 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2] in a final volume of 50 μL. Enzyme was first incubated with the inhibitor for 5 min before the reaction was initiated with substrate; the most potent inhibitor, compound 6, was also evaluated after incubation for 30 and 60 min to assess the possibility of time-dependent inhibition. After 30 min, reactions were quenched by adding 100 μM M344 (Sigma-Aldrich) and the appropriate Fluor-de-Lys developer (BML-KI105, Enzo Life Sciences, 50 μL). Fluorescence was measured after 45 min using a Fluoroskan II plate reader (excitation at 355 nm, emission at 460 nm).
Affinity data for this assay
 

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Last update November 1, 2007
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