Recombinant Human Dihydroorotate Dehydrogenase Protein, CF Summary
Thr31 - Arg395
with N-terminal Met and 6-His tag
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- 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
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhDHODH to 0.4 µg/mL in Assay Buffer.
- Prepare Substrate Mixture containing 2 mM L-Dihydroorotic acid, 0.2 mM Decylubiquinone and 0.12 mM DPIP in Assay Buffer.
- 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.
- Immediately read plate in kinetic mode for 5 minutes at 600 nm (absorbance).
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x (-1) x well volume (L) x 1012 pmol/mol|
|ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)|
*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
Recombinant Human DHODH (Catalog # 10062-DD) is measured byits reduction of 2,6-dichloroindophenol during oxidation of dihydroorotate.
2 μg/lane of Recombinant Human DHODH was resolved with SDS-PAGE underreducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Bluestaining, showing a band at 41 kDa.
Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme in the pyrimidine de novo pathway that converts dihydroorotate to orotate. DHODHs have a monomeric structure composed of a large C-terminal a/b barrel and a small N-terminal helical domain (1). There are two main classes of DHODHs based on similarity, preference of substrate, and subcellular location (1,2). Within Class 2, structural differences can be exploited for selective targeting (3,4). Human DHODH belongs to Class 2 and is a monomeric, mitochondrial, FMN-dependent enzyme (2). It is ubiquitously expressed in most tissue. As DHODH catalyzes de novo pyrimidine synthesis for synthesis of DNA/RNA essential to rapidly proliferating cells, DHODH is currently a target for treatment of cancer (5-8). It has also been successfully targeted in treatment for rheumatoid arthritis (9-10), multiple sclerosis (11-12), viral infection (13-14), microbial infectious diseases such as malaria (4, 15) and gastrointestinal disease (3, 16), and antifungal infection (17). Mutations in the DHODH resulting in functional defects cause Miller syndrome, also known as postaxial acrofacial dystosis syndrome (POADS) (18).
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