Recombinant Human POR Protein, CF

Product Discontinued

Recombinant Human POR Protein, CF Summary

• Details of Functionality: Measured by the reduction of cytochrome c using NADPH as the cofactor. The specific activity is >6,500 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human POR/Cytochrome P450 Reductase protein
  Arg45-Ser677, with N-terminal amino acids WAGALA and 6-His tag
• Accession #: P16435.2
• N-terminal Sequence: N-terminal Trp & P16435.2 Ser62
• Protein/Peptide Type: Recombinant Enzymes
• Gene: POR
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 73 kDa & 70 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 65-80 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Assay Buffer: 0.3 M KH₂PO₄, pH 8.0
  • Recombinant Human POR/Cytochrome P450 Reductase (rhPOR) (Catalog # 6340-PR)
  • Substrate: beta -NADPH (Sigma, Catalog # N7505), 10 mM stock in deionized water (8.33 mg/mL)
  • Cytochrome C, Bovine heart (Sigma, Catalog # C3131), 2 mg/mL stock in deionized water
  • 96 well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhPOR to 0.2 ng/μL in Assay Buffer.
  2. Combine 480 μL of Assay Buffer, 500 μL of 2 mg/mL Cytochrome C, and 20 μL of 10 mM beta -NADPH.
  3. Load 50 μL of 0.2 ng/μL rhPOR and start the reaction by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL Substrate Mixture.
  4. Read in kinetic mode for 6 minutes with 1 minute lag time at an absorbance of 550 nm.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 21100 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhPOR: 0.010 μg
  • Cytochrome C: 0.5 mg/ml
  • beta -NADPH: 0.1 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human POR Protein, CF

• CPR
• CYPOR
• CYPORFLJ26468
• Cytochrome P450 Reductase
• DKFZp686G04235
• EC 1.6.2.4
• NADPH--cytochrome P450 reductase
• NADPH-dependent cytochrome P450 reductase
• P450 (cytochrome) oxidoreductase
• P450R
• POR

Background

NADPH-Cytochrome P450 Reductase (P450R) is an essential component of the cytochrome P450 monooxygenase system of eukaryotic cells (1). P450R is anchored in the endoplasmic reticulum membrane with its catalytic domain residing in the cytosol. P450R is a flavoprotein, containing one molecule each of FMN and FAD, which are essential for the transfer of electrons from NADPH to the cytochromes P450 (2). This reduction is necessary for cytochromes P450 to perform each cycle of oxidation. P450R is also capable of transferring electrons to cytochrome b₅, heme oxygenase, the fatty acid elongation system, and other proteins. Mutations of P450R can result in disordered steroidogenesis and Antley-Bixler syndrome.

1. Philips, A.H. and R.G. Langdon (1962) J. Biol. Chem. 237:2652.
2. Iyanagi, T. and H.S. Mason (1973) Biochemistry 12:2291.
3. Flueck C.E. et al. (2004) Nat. Genet. 36:228.

Bioinformatics

• Gene Symbol: POR
• Uniprot:
  • P16435.2()