Recombinant Human HO-2/HMOX2 Protein, CF

Catalog #: 3170-HM Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
3170-HM-010

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Human HO-2/HMOX2 Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

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

Activity

Measured by its ability to oxidize hemin to biliverdin. The specific activity is >3.5 pmol/min/μg, as measured under the described conditions.

Source

E. coli-derived human HO-2/HMOX2 protein
Ser2-Leu291, with a C-terminal 6-His tag

Accession #

P30519

N-terminal Sequence
Analysis

Ser2

Predicted Molecular Mass

34 kDa

SDS-PAGE

31 & 35 kDa, reducing conditions

Product Datasheets

3170-HM

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.

3170-HM

Formulation	Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping	The product is shipped with dry ice or equivalent. 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, -70 °C as supplied. 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Assay Buffer: 50 mM Tris, pH 7.5
Recombinant Human HO‑2/HMOX2 (rhHO‑2) (Catalog # 3170-HM)
Recombinant Human POR/Cytochrome P450 Reductase (rhPOR) (Catalog # 6340-PR)
Recombinant Human Biliverdin Reductase A/BLVRA (rhBLVRA) (Catalog # 6454-BR)
Catalase (Sigma, Catalog # C30), 100,000 U/mL stock
Hemin (Sigma, Catalog # H9039), 10 mM stock in DMSO
Bovine Serum Albumin (BSA), 100 mg/mL stock in deionized water
beta -Nicotinamide adenine 2'-phosphate reduced tetrasodium salt hydrate (NADPH) (Sigma, Catalog # N7505), 20 mM stock in deionized water
96-well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Dilute Hemin to 1 mM in Assay Buffer.
Prepare Reaction Mixture containing 60 µM Hemin, 4 mg/mL BSA, 1000 U/mL catalase, 80 µg/mL rhPOR, and 80 µg/mL rhBLVRA in Assay Buffer.
Prepare 80 µg/mL rhHO-2 in Assay Buffer.
Dilute NADPH to 1 mM in Assay Buffer.
In a clear plate, load 25 µL dilute rhHO-2 and add 25 µL Reaction Mixture. Include an enzyme blank containing 25 µL Assay Buffer and 25 µL Reaction Mixture.
Start the reaction by adding 50 µL 1 mM NADPH to all wells used.
Read absorbance at 468 nm (bottom read) 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 Substrate Blank
     **Using the extinction coefficient 43500 M^-1cm^-1
     ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD Per Reaction:

rhHO-2: 2 µg
rhPOR: 2 µg
rhBLVRA: 2 µg
Catalase: 250 U/mL
Hemin: 15 µM
BSA: 1 mg/mL
NADPH: 0.5 mM

Reconstitution Calculator

Background: HO-2/HMOX2

Heme oxygenase (HO) is the rate limiting enzyme in heme catabolism (1). It cleaves heme to biliverdin, carbon monoxide, and iron; biliverdin is subsequently converted by biliverdin reductase to bilirubin. The mechanism of HO is unique in that heme serves as both the substrate of the enzyme and as the prosthetic group for the activation of iron-bound O₂. HO activity is highest in spleen where senescent erythrocytes are sequestered and destroyed. Two isoforms, HO-1 and HO-2, are expressed in most tissues. HO-1 is an inducible enzyme in response to heavy metals, oxidative stress, cytokines, and many drugs (2). HO-2 is constitutively expressed in the brain and testes. HO-1 is expressed mainly in spleen, liver, and kidney. In addition to its activity in the heme degradation pathway, HO-2 potentially serves as an oxygen sensor through its heme regulating motifs, which have the core sequence of Cys-Pro (3).

References

Nader, G. (2008) Pharmacol. Rev. 60:79.
Otterbein, L.E. and A.M.K. Choi (2000) Am. J. Physiol. Lung Cell Mol. Physiol. 279:1029.
Yi. L. et al. (2009) J. Biol. Chem. 284:20556.

Long Name

Heme Oxygenase 2

Entrez Gene IDs

3163 (Human); 15369 (Mouse); 79239 (Rat)

Alternate Names

heme oxygenase (decycling) 2; heme oxygenase 2; HMOX2; HO2; HO-2; HO-2EC 1.14.99.3