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Assay Method Information

Assay Name:  Enzyme Inhibition on the Conversion of E2 to E1 by 17beta-HSD1
Description:  For steady-state kinetic study of hybrid inhibitors, a Fluorolog 3 instrument was used to monitor the fluorescent signal of NADPH formed during estradiol oxidation, through which the substrate conversion is monitored. The excitation wavelength was 340 nm, and the emission wavelength was 460 nm. A standard curve monitoring the increase of fluorescence (cps) caused by an increase of NADPH revealed that the formation of 1 uM NADPH corresponds to a 316,000 cps increase. The Km for E2 in the absence of EM-1745, and four apparent Km values in the presence of 0.5, 2, 4, and 7 nM EM-1745 were obtained. Ki was further determined by a plot of Km vs. the inhibitor concentration.
Affinity data for this assay
 

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Last update November 1, 2007
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