Recombinant Human Dihydroorotate Dehydrogenase Protein, CF

• Assay Buffer: 50 mM Tris, 150 mM KCl, 0.1% Triton^® X-100, pH 8.0
  
• Recombinant Human DHODH His-tag (rhDHODH) (Catalog # 10062-DD)
  
• L-Dihydroorotic acid (Sigma, Catalog # D7128), 40 mM stock in DMF
  
• Decylubiquinone (Sigma, Catalog # D7911), 20 mM stock in DMSO
  
• 2,6-Dichloroindophenol sodium salt hydrate (DPIP) (Sigma, Catalog # D1878), 2 mM in Absolute Ethanol
  
• 96-well Clear Plate (Catalog # DY990)
  
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhDHODH to 0.4 µg/mL in Assay Buffer.
2. Prepare Substrate Mixture containing 2 mM L-Dihydroorotic acid, 0.2 mM Decylubiquinone and 0.12 mM DPIP in Assay Buffer.
3. Load 50 µL of 0.4 µg/mL rhDHODH to plate, and start the reaction by adding 50 µL of Substrate Mixture.  Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
4. Immediately read plate in kinetic mode for 5 minutes at 600 nm (absorbance).
5. Calculate specific activity:

*Adjusted for Substrate Blank
**Using the extinction coefficient 21,000 M^-1 cm^-1.
***Using the path correction 0.32 cm.
Note: the output of many spectrophotometers is in mOD.

• rhDHODH: 0.02 µg
• L-Dihydroorotic acid: 1 mM
• Decylubiquinone: 0.1 mM
• DPIP: 0.06 mM