Recombinant Human Biliverdin Reductase A/BLVRA Protein, CF

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

Recombinant Human Biliverdin Reductase A/BLVRA Protein, CF Summary

• Details of Functionality: Measured by the reduction of biliverdin IX alpha to bilirubin. The specific activity is >450 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Biliverdin Reductase A/BLVRA protein
  Glu6-Ser294, with an N-terminal Met and C-terminal 6-His tag
• Accession #: P53004
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: BLVRA
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 34 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: 38 kDa, 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, Brij and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 100 mM Tris, pH 8.5
  • Recombinant Human Biliverdin Reductase A/BLVRA (rhBLVRA) (Catalog # 6454-BR)
  • Bovine Serum Albumin (BSA), 100 mg/mL in deionized water
  • Biliverdin (Frontier Scientific, Catalog # B655-9), 1 mM in DMSO
  • beta -NADPH (Sigma, Catalog # N7505), 10 mM in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhBLVRA to 3 ng/μL in Assay Buffer.
  2. Dilute BSA to 3 mg/mL in Assay Buffer.
  3. Dilute Biliverdin to 60 μM in Assay Buffer.
  4. Combine equal volumes of diluted rhBLVRA, BSA, and Biliverdin to form the reaction mixture. As a control, use Assay Buffer in place of rhBLVRA.
  5. Dilute beta -NADPH to 200 μM in Assay Buffer.
  6. Load 50 μL of reaction mixture and control into the microplate and start the reaction by adding 50 μL of 200 μM beta -NADPH.
  7. Read at an absorbance of 468 nm in kinetic mode for 5 minutes.
  8. 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 Control
  **Using the extinction coefficient 55000 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhBLVRA: 0.05 μg
  • Biliverdin: 10 μM
  • beta -NADPH: 100 μM

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 Biliverdin Reductase A/BLVRA Protein, CF

• Biliverdin Reductase A
• Biliverdin-IX alpha-reductase
• BLVR
• BLVRA
• BVR A
• BVR
• BVRA
• EC 1.3.1.24

Background

The clearance of heme in mammals is a two-step process starting with the conversion of heme to biliverdin by heme oxygenase, followed by reduction of biliverdin to bilirubin by biliveredin reductase. Biliverdin Reductase A (BLVRA) converts the alpha  isomer of biliverdin IX, which constitutes 95‑97% of total biliverdin isomers in adults, to bilirubin IX-alpha (1). Although bilirubin is known to be a toxic pigment that needs to be excreted, it is also a physiological antioxidant (2). Therefore BLVRA enables continuous protection of cells against oxidative stress. BLVRA is a cytosolic protein that is highly expressed in the liver, but is also present in most tissues. The two N‑terminal residues are a pro-sequence that is missing in the mature protein (3). The reduction of biliverdin by BLVRA is coupled to oxidation of pyridine nucleotide co‑factors NADH and NADPH with distinct pH optima, 6.7 for NADH and 8.7 for NADPH (4). BLVRA has many functions independent of its reductase activity. It is also a dual-specificity (Ser/Thr + Tyr) protein kinase (5) and an activator of protein kinase C bII (6) and ERK (7). It functions as a transcription factor regulating heme oxygenase gene expression (8).

1. Yamaguchi, T. et al. (1994) J. Biol. Chem. 269:24343.
2. Stocker, R. et al. (1987) Science 235:1043.
3. Fakhrai, H. and M.D. Maines. (1992) J. Biol. Chem. 267:4023.
4. Maines, M. D. et al. (1996) Eur. J. Biochem. 235:372.
5. Kapitulnik, J. and M.D. Maines. (2009) Trends Pharmacol. Sci. 30:129.
6. Maines, M.D. et al. (2007) J. Biol. Chem. 282:8110.
7. Lerner-Marmarosh, N. et al. (2008) Proc. Natl. Acad. Sci. USA. 105:6870.
8. Tudor, C. et al. (2008) Biochem. J. 413:405.

Bioinformatics

• Gene Symbol: BLVRA
• Uniprot:
  • P53004()