Recombinant Human HO-1/HMOX1 Protein, CF Summary
Product Specifications
Met1-Thr261 (Phe33Leu), with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
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.
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.
|
Assay Procedure
- 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
- 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-1 in Assay Buffer.
- Dilute NADPH to 1 mM in Assay Buffer.
- 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.
- 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 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
Reconstitution Calculator
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.
- Kappas, A. (2008) Pharmacol. Rev. 60:79.
- Otterbein, L.E. and Choi, A.M.K. (2000) Am. J. Physiol. Lung Cell. Mol. Physiol. 279:1029.
- Grochot-Przeczek, A. et al. (2012) Clin. Sci. (Lond) 122:93.
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