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
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
Publications
Read Publication using 6568-BR in the following applications:
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
Dilute rhBLVRB to 40 ng/μL in Assay Buffer.
Prepare a Reaction Mixture by combining FMN and beta -NADPH in Assay Buffer to a concentration of 400 μM for each.
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.
Read at an absorbance of 340 nm in kinetic mode for 5 minutes (Note: readings will be negative).
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 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).
Pereira, P.J. et al. (2001) Nat. Struct. Biol. 8:215.
Chikuba, K. et al. (1994) Biochem. Biophys. Res. Commun. 198:1170.
Shalloe, F. et al. (1996) Biochem. J. 316:385.
Cunningham, O. et al. (2000) Biochem. J. 345:393.
Yamaguchi, T. et al. (1994) J. Biol. Chem. 269:24343.
Cunningham, O. et al. (2000) J. Biol. Chem. 275:19009.
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