Recombinant Human Biliverdin Reductase B/BLVRB Catalog Number: 6568-BR DESCRIPTION Source E. coli­derived human Biliverdin Reductase B/BLVRB protein Ala2­Gln206, with an N­terminal Met and 6­His tag Accession # P30043 N­terminal Sequence N­terminus confirmed by detection of His tag using Western analysis. Analysis Predicted Molecular 23 kDa Mass SPECIFICATIONS SDS­PAGE 23­25 kDa, reducing conditions Activity 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. Endotoxin Level <1.0 EU per 1 μg of the protein by the LAL method. Purity >95%, by SDS­PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Brij and Glycerol. See Certificate of Analysis for details. Activity Assay Protocol Materials l Assay Buffer: 100 mM Sodium Acetate, pH 5.0 l Recombinant Human Biliverdin Reductase B/BLVRB (rhBLVRB) (Catalog # 6568­BR) l Riboflavin 5’­monophosphate sodium salt dihydrate (FMN) (Sigma, Catalog # F6750), 10 mM in deionized water l β­Nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt (β­NADPH) (Sigma, Catalog # N7505), 10 mM in deionized water l UV Plate (Costar, Catalog # 3635) l Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent Assay 1. Dilute rhBLVRB to 40 ng/μL in Assay Buffer. 2. Prepare a Reaction Mixture by combining FMN and β­NADPH 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: Adjusted V * (OD/min) x ­1 x well volume (L) x 1012 pmol/mol Specific Activity (pmol/min/µg) = max ext. coeff** (M­1cm­1) x path corr.*** (cm) x amount of enzyme (µg) *Adjusted for Substrate Blank **Using the extinction coefficient 6270 M­1cm­1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD. Final Assay Per Well: Conditions l rhBLVRB: 2 μg l FMN: 200 μM l β­NADPH: 200 μM PREPARATION AND STORAGE Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. Stability & Storage Use a manual defrost freezer and avoid repeated freeze­thaw cycles. l 6 months from date of receipt, ­20 to ­70 °C as supplied. l 3 months, ­20 to ­70 °C under sterile conditions after opening. BACKGROUND Clearance of heme in mammals is a two­step 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 β 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 NADPH­dependent reduction for a variety of flavins, such as riboflavin, FAD or FMN and met­hemoglobin (3, 4). BLVRB is structurally distinct from BLVRA. In contrast to BLVRA, which prefers the biliverdin α isomer but could also use the β isomer as substrate, BLVRB is specific for the β isomer (5, 6). References: 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. Rev. 5/11/2018 Page 1 of 1 .