| Assay Method Information | |
| | Method for Testing Activities of Compounds at Enzyme Level |
| Description: | Principles of activity test at enzyme level: firstly, dihydroorotate (DHO) is oxidatively dehydrogenated by DHODH to form orotic acid (Orotate, OA), accompanied by reduction of Flavin mononucleotide (FMN) into reduced flavin mononucleotide (FMNH2) by accepting 2H+ and 2e−, then coenzyme Q (CoQ) as hydrogen acceptor accepting electron and proton from FMNH2 is reduced into reduced coenzyme Q (CoQH2), reduced coenzyme Q transfers electrons to a chromogenic substrate, diclofenac sodium salt (DCIP), and finally DCIP was reduced. DCIP shows maximum absorption at 600 nm, while the reduced DCIP does not show absorption at 600 nm. The degree to which the substrate DHO is oxidized can be judged based on the degree of decrease in absorbance. The degree of oxidation of the substrate DHO per unit time is the initial rate of the enzymatic reaction. Upon addition of a inhibitor, the initial rate of the enzymatic reaction is reduced.The purified DHODH was diluted to 10 nM with a test buffer (50 mM HEPES, pH 8.0, 150 mM KCl, 0.1% Triton X-100), and coenzyme Q and DCIP were added to a final concentration of 100 μM and 120 μM, respectively, mixed well, added into a 96-well plate and incubated for 5 min at room temperature. Substrate DHO was added to start the reaction with a DHO final concentration of 500 μM. The absorbance was read at 600 nm using a BioTek microplate reader and read every 30 s for 6 min. For inhibitor activity test, different concentrations of inhibitors were added into the above reaction system, the initial rate of enzymatic reaction when no inhibitor was added was V0, and the initial rate of enzymatic reaction after an inhibitor was added was Vi, and the inhibition rate of a compound was calculated by the formula (1−Vi/V0)×100%. For calculating IC50 of a compound, Origin 8.0 was used to test the inhibition rate at least 8 concentrations. In the experimental procedure, Brequinar (purchased from Sigma-Aldrich Reagent) was used as a positive control and at least three parallels were set in each experiment. |
| Affinity data for this assay | |
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