Recombinant Human POR/Cytochrome P450 Reductase Protein, CF

Recombinant Human POR His-tag (Catalog # 6340-PRB) is measured by the reduction of cytochrome c using NADPH as the cofactor.

2 μg/lane of Recombinant Human POR/Cytochrome P450 Reductase His-tag Protein (Catalog # 6340-PRB) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 63-69 kDa, under reducing conditions.

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human POR/Cytochrome P450 Reductase Protein, CF Summary

• Additional Information: His-tag
• Details of Functionality: Measured by the reduction of cytochrome c using NADPH as the cofactor. The specific activity is >1800 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human POR/Cytochrome P450 Reductase protein
  Ser62-Ser677 with an N-terminal Met and 6-His tag
• Accession #: P16435.2
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 71 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: 63-69 kDa, under reducing conditions.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 0.3 M Potassium Phosphate, pH 8.0
  • Recombinant Human POR/Cytochrome P450 Reductase His-tag (rhPOR) (Catalog # 6340-PRB)
  • Substrate: beta -NADPH, 10 mM stock in deionized water
  • Cytochrome C, Bovine heart, 2 mg/mL stock in deionized water
  • Clear 96 well Plate (Catalog # DY990)
  • Plate Reader with Absorbance Read Capability
  1. Dilute rhPOR to 0.2 μg/mL in Assay Buffer.
  2. Prepare a Substrate Mixture containing 200 µM beta -NADPH and 1 mg/mL Cytochrome C in Assay Buffer.
  3. Load 50 μL of 0.2 μg/mL 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: 100 μM

Notes

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

Alternate Names for Recombinant Human POR/Cytochrome P450 Reductase 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), or POR, 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. The protein is composed of four domains: an N-terminal FMN‑binding domain, a connecting domain, and C-terminal FAD- and NADPH-binding domains (2). The flavoprotein P450R contains one molecule each of FMN and FAD, which are essential for the transfer of electrons from NADPH to the P450 cytochromes (3). This reduction is necessary for cytochromes P450 to perform each cycle of oxidation. P450R plays a vital role in metabolism and detoxification of xenobiotic and endobiotic compounds (4). P450R is also capable of transferring electrons to cytochrome b5, heme oxygenase, the fatty acid elongation system, and other proteins (4). Mutations of P450R can result in disordered steroidogenesis and bone defects in Antley-Bixler syndrome (5, 6) and expression of P450R has been correlated with patient outcome in breast cancer (7).

1. Philips, A.H. and R.G. Langdon (1962) J. Biol. Chem. 237:2652.
2. Wang, M. et al. (1997) Proc. Natl. Acad. Sci. 94:8411.
3. Iyanagi, T. and H.S. Mason (1973) Biochemistry 12:2291.
4. Iyanagi, T. (2007) Int. Rev. Cytol. 260:35.
5. Flueck C.E. et al. (2004) Nat. Genet. 36:228.
6. Polusani, S.R. et al. (2011) Biochem. Biophys. Res. Commun. 411:490.
7. Pederson, M.H. et al. (2019) Int. J. Cancer 144:631.

Bioinformatics

• Uniprot:
  • P16435.2()