Active NADH Dehydrogenase, Quinone 1 (NQO1)

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), also known as DT-diaphorase, is a widely-distributed FAD-dependent flavoprotein that promotes 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes. As a result it prevents the one electron reduction of quinones that results in the production of radical species. NQO1 is a highly-inducible enzyme that is regulated by the Keap1/Nrf2/ARE pathway. The increase and decrease of NQO1 levels are associated with decreased and increased susceptibilities to oxidative stress, respectively. Thus, NQO1 is a marker cytoprotective enzyme in oxidative stress. Independently of its catalytic function, NQO1 plays a role in regulating the proteosomal degradation of p53, p73a, and p33. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation in a ubiquitin independent pathway. The activity assay of recombinant rat NQO1 was measured by its ability to oxidize the substrate resazurin to resorufin. The rrNQO1 was diluted to 0.05 ug/ml in the assay buffer 50 mM HEPES, 0.2 M NaCl, 5 µM FAD, 0.05% Tween® 20, pH 7.5. 50 ul 0.05 ug/ml rhNQO1 was added into the microplate and start the reaction by adding 50 µl substrate mixture of 400 uM beta-NADH and 20 uM resazurin which was diluted in assay buffer. Read at excitation and emission wavelengths of 540 nm and 585 nm (top read), respectively, in kinetic mode for 5 minutes. The specific activity of recombinant rat NQO1 is >45000 pmol/min/µg.