Azoreductase/NQO1 Protein, Human (His)

Azo reductase/NQO1 protein is a flavin-containing quinone reductase that uses NADH or NADPH to catalyze the two-electron reduction of quinone to hydroquinone. It regulates cellular redox status by detoxifying quinones and reducing plasma membrane redox components. Azoreductase/NQO1 Protein, Human (His) is the recombinant human-derived Azoreductase/NQO1 protein, expressed by E. coli , with N-His labeled tag.

Azoreductase/NQO1 protein is a flavin-containing quinone reductase that facilitates the two-electron reduction of quinones to hydroquinones, utilizing either NADH or NADPH as electron donors. Operating through a ping-pong kinetic mechanism, the electrons are sequentially transferred from NAD(P)H to the flavin cofactor and subsequently to the quinone, effectively bypassing the generation of semiquinone and reactive oxygen species. This enzymatic activity plays a crucial role in regulating cellular redox balance by detoxifying quinones. Azoreductase/NQO1 serves as a superoxide scavenger, preventing hydroquinone oxidation and supporting antioxidant defense mechanisms. Moreover, it participates in the activation of quinones, generating redox-reactive hydroquinones with potential antitumor properties through DNA cross-linking. Notably, the protein acts as a gatekeeper for the core 20S proteasome, interacting with tumor suppressors TP53 and TP73 in a NADH-dependent manner to inhibit their ubiquitin-independent degradation during oxidative stress.

Measured by its ability to catalyze 10 µM Resazurin and 200 µM NADH. The specific activity is >15,000 pmol/min/μg.

Materials
Assay buffer: 50 mM HEPES, 0.2 M NaCl, 5 μM FAD, 0.05% Tween? 20, pH 7.5
Azoreductase/NQO1 Protein, Human (His) (HY-P74405)
Substrates: Resazurin (HY-111391), NADH
Standard: Resorufin (HY-123533A)

Procedure
1. Dilute Resorufin with assay buffer to the following concentrations: 0, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 μM.
2. Add 100 μL of each concentration of standard into wells.
3. Measure the Excitation/emission values at 540 nm and 585 nm using a microplate reader.
4. Plot the standard curve with the amount of standard on the x-axis and the measured RFU on the y-axis to obtain the standard curve equation.
5. First, reconstitute the Human NQO1 protein with pure water to 100 μg/mL, then dilute it to 0.0075 μg/mL with assay buffer.
6. Dilute NADH to 800 μM in the assay buffer.
7. Dilute Resazurin to 40 μM in assay buffer.
8. Mix equal volumes of diluted NADH and Resazurin to prepare the substrate mixture. Use immediately after mixing.
9. Add 50 μL of 0.0075 μg/mL Human NQO1 into the plate, then add 50 μL of substrate mixture to start the reaction. For the blank group, add 50 μL of assay buffer and 50 μL of substrate mixture.
10. Measure the Excitation/emission values at 540 nm and 585 nm using a microplate reader.
11. Calculate Specific Activity:

Human

E. coli

N-10*His

P15559-1 (M1-K274)

1728  [NCBI]

Full Length of Isoform-1

MVGRRALIVLAHSERTSFNYAMKEAAAAALKKKGWEVVESDLYAMNFNPIISRKDITGKLKDPANFQYPAESVLAYKEGHLSPDIVAEQKKLEAADLVIFQFPLQWFGVPAILKGWFERVFIGEFAYTYAAMYDKGPFRSKKAVLSITTGGSGSMYSLQGIHGDMNVILWPIQSGILHFCGFQVLEPQLTYSIGHTPADARIQILEGWKKRLENIWDETPLYFAPSSLFDLNFQAGFLMKKEVQDEEKNKKFGLSVGHHLGKSIPTDNQIKARK

Approximately 33 kDa

• 
  ≥ 90%, as determined by reducing SDS-PAGE.

Lyophilized powder

1.Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4, 5% trehalose, 5% mannitol, 0.01% Tween 80.
2.Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4, 8% trehalose.
Please refer to the lot-specific COA for specific buffer information.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.