Recombinant Human Biliverdin Reductase B/BLVRB Protein, CF

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

Recombinant Human Biliverdin Reductase B/BLVRB Protein, CF Summary

• Details of Functionality: Measured by the reduction of riboflavin 5'-monophosphate (FMN) using NADPH as the cofactor. The specific activity is >225 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Biliverdin Reductase B/BLVRB protein
  Ala2-Gln206, with an N-terminal Met and 6-His tag
• Accession #: P30043
• N-terminal Sequence: N-terminus confirmed by detection of His tag using Western analysis.
• Protein/Peptide Type: Recombinant Enzymes
• Gene: BLVRB
• 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: 23 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: 23-25 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 Sodium Acetate, pH 5.0
  • Recombinant Human Biliverdin Reductase B/BLVRB (rhBLVRB) (Catalog # 6568-BR)
  • Riboflavin 5’-monophosphate sodium salt dihydrate (FMN) (Sigma, Catalog # F6750), 10 mM in deionized water
  • beta -Nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt ( beta -NADPH) (Sigma, Catalog # N7505), 10 mM in deionized water
  • UV Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhBLVRB to 40 ng/μL in Assay Buffer.
  2. Prepare a Reaction Mixture by combining FMN and beta -NADPH in Assay Buffer to a concentration of 400 μM for each.
  3. Load 50 μL of 40 ng/μL rhBLVRB into the microplate, and start the reaction by adding 50 μL of Reaction Mixture. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Reaction Mixture.
  4. Read at an absorbance of 340 nm in kinetic mode for 5 minutes (Note: readings will be negative).
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x -1 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 6270 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD. Per Well:
  • rhBLVRB: 2 μg
  • FMN: 200 μM
  • beta -NADPH: 200 μM

Notes

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

Alternate Names for Recombinant Human Biliverdin Reductase B/BLVRB Protein, CF

• biliverdin reductase B (flavin reductase (NADPH))
• Biliverdin Reductase B
• Biliverdin-IX beta-reductase
• BLVRB
• BVRB
• BVR-B
• EC 1.3.1.24
• EC 1.5.1.30
• flavin reductase (NADPH)
• flavin reductase
• FLR
• FLRBVRB
• FR
• GHBP
• Green heme-binding protein
• MGC117413
• NADPH-dependent diaphorase
• NADPH-flavin reductase
• SDR43U1
• short chain dehydrogenase/reductase family 43U, member 1

Background

Clearance of heme in mammals is a two-step process starting with conversion of heme to biliverdin by heme oxygenase, followed by reduction of biliverdin to bilirubin by bilivredin reductase. Biliverdin IX b reductase (BLVRB) converts the  beta  isomer of biliverdin IX to bilirubin IX b, which constitutes 87% of total bilirubin in fetal bile. Therefore BLVRB is especially important for fetal heme metabolism and clearance (1). BLVRB is a cytoplasmic enzyme expressed at high levels in erythrocytes and liver, but is present in other tissues (2). The enzyme is identical to flavin reductase, which is an oxidoreductase that catalyses the NADPH-dependent reduction for a variety of flavins, such as riboflavin, FAD or FMN and met‑hemoglobin (3, 4). BLVRB is structurally distinct from BLVRA. In contrast to BLVRA, which prefers the biliverdin alpha  isomer but could also use the  beta  isomer as substrate, BLVRB is specific for the beta  isomer (5, 6).

1. Pereira, P.J. et al. (2001) Nat. Struct. Biol. 8:215.
2. Chikuba, K. et al. (1994) Biochem. Biophys. Res. Commun. 198:1170.
3. Shalloe, F. et al. (1996) Biochem. J. 316:385.
4. Cunningham, O. et al. (2000) Biochem. J. 345:393.
5. Yamaguchi, T. et al. (1994) J. Biol. Chem. 269:24343.
6. Cunningham, O. et al. (2000) J. Biol. Chem. 275:19009.

Publications for Biliverdin Reductase B/BLVRB (6568-BR)(1)

Publication using 6568-BR

• Rees M, Seashore-Ludlow B, Cheah J, Adams D, Price E, Gill S, Javaid S, Coletti M, Jones V, Bodycombe N, Soule C, Alexander B, Li A, Montgomery P, Kotz J, Hon C, Munoz B, Liefeld T, Dancik V, Haber D, Clish C, Bittker J, Palmer M, Wagner B, Clemons P, Shamji A, Schreiber S Correlating chemical sensitivity and basal gene expression reveals mechanism of action. Nat Chem Biol, 2015-12-14;12(2):109-16. 2015-12-14 [PMID: 26656090] (Bioassay, Human)

Bioinformatics

• Gene Symbol: BLVRB
• Uniprot:
  • P30043()