Recombinant Human HO-1/HMOX1 Protein, CF

R&D Systems | Catalog # 3776-HM

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human HO-1/HMOX1 Protein (3776-HM)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P09601

Applications

Enzyme Activity
View all HO-1/HMOX1/HSP32 Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human HO-1/HMOX1/HSP32 protein
Met1-Thr261 (Phe33Leu), with a C-terminal 6-His tag

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.

N-terminal Sequence Analysis

Met1

Predicted Molecular Mass

31 kDa

SDS-PAGE

29-31 kDa, reducing conditions

Activity

Measured by its ability to oxidize hemin to biliverdin.
The specific activity is >5 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

3776-HM
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl 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.

Background: HO-1/HMOX1/HSP32

Heme oxygenase (HMOX) is the rate limiting enzyme in heme catabolism (1). It cleaves heme to biliverdin, carbon monoxide, and iron. The biliverdin is subsequently converted to bilirubin by biliverdin reductase. The mechanism of HMOX is unique in that heme serves as the substrate of the enzyme and as the prosthetic group for the activation of iron-bound O2. HMOX activity is highest in spleen where senescent erythrocytes are sequestered and destroyed. Two isoforms, HMOX1 and HMOX2, are expressed in most tissues. HMOX1 is an inducible enzyme in response to heme, heavy metals, oxidative stress, cytokines, and many drugs (2). Whereas HMOX2 displays a constitutive expression. HMOX1 is expressed mainly in spleen, liver, and kidney, and HMOX2 is prominently expressed in the brain and testes. The increased expression of HMOX1 levels is related to a variety of pathological states, where it functions as a cytoprotective molecule through its by‑products (3). HMOX1 also plays important roles in the regulation of cell proliferation, differentiation, and apoptosis.

References

  1. Kappas, A. (2008) Pharmacol. Rev. 60:79.
  2. Otterbein, L.E. and Choi, A.M.K. (2000) Am. J. Physiol. Lung Cell. Mol. Physiol. 279:1029.
  3. Grochot-Przeczek, A. et al. (2012) Clin. Sci. (Lond) 122:93.

Long Name

Heme Oxygenase 1

Alternate Names

HMOX1, HO1, HSP32

Entrez Gene IDs

3162 (Human); 24451 (Mouse); 15368 (Rat)

Gene Symbol

HMOX1

UniProt

P09601

Additional HO-1/HMOX1/HSP32 Products

Product Documents for Recombinant Human HO-1/HMOX1 Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human HO-1/HMOX1 Protein, CF

For research use only

Citations for Recombinant Human HO-1/HMOX1 Protein, CF

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Protocols

View specific protocols for Recombinant Human HO-1/HMOX1 Protein, CF (3776-HM):

Materials
  • Assay Buffer: 50 mM Tris, pH 7.5
  • Recombinant Human HO‑1/HMOX1/HSP32 (rhHO-1) (Catalog # 3776-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)
  • 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), 10 mM stock in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute Hemin to 1 mM in Assay Buffer.
  2. 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.
  3. Prepare 80 µg/mL rhHO-1 in Assay Buffer.
  4. Dilute NADPH to 1 mM in Assay Buffer.
  5. In a clear plate, load 25 µL dilute rhHO-1 and add 25 µL Reaction Mixture. Include an enzyme blank containing 25 µL Assay Buffer and 25 µL Reaction Mixture.
  6. Start the reaction by adding 50 µL 1 mM NADPH to all wells used.
  7. Read absorbance at 468 nm (bottom read) in kinetic mode for 5 minutes.
  8. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (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-1: 2 µg
  • rhPOR: 2 µg
  • rhBLVRA: 2 µg
  • Catalase: 250 U/mL
  • Hemin: 15 µM
  • BSA: 1 mg/mL
  • NADPH: 0.5 mM

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