Recombinant Human Biliverdin Reductase A/BLVRA Protein, CF

Catalog #: 6454-BR Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
6454-BR-020

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human Biliverdin Reductase A/BLVRA Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

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
Analysis

Met

Predicted Molecular Mass

34 kDa

SDS-PAGE

38 kDa, reducing conditions

Product Datasheets

6454-BR

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

6454-BR

Formulation	Supplied as a 0.2 μm filtered solution in Tris, NaCl, Brij and Glycerol.
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 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.

Assay Procedure

Materials

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

Dilute rhBLVRA to 3 ng/μL in Assay Buffer.
Dilute BSA to 3 mg/mL in Assay Buffer.
Dilute Biliverdin to 60 μM in Assay Buffer.
Combine equal volumes of diluted rhBLVRA, BSA, and Biliverdin to form the reaction mixture. As a control, use Assay Buffer in place of rhBLVRA.
Dilute beta -NADPH to 200 μM in Assay Buffer.
Load 50 μL of reaction mixture and control into the microplate and start the reaction by adding 50 μL of 200 μM beta -NADPH.
Read at an absorbance of 468 nm in kinetic mode for 5 minutes.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (OD/min) x well volume (L) x 10¹² 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

Reconstitution Calculator

Background: Biliverdin Reductase A/BLVRA

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).

References