Recombinant P. heparinus Heparinase III Protein, CF

R&D Systems | Catalog # 6145-GH

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Key Product Details

  • R&D Systems E. coli-derived Recombinant P. heparinus Heparinase III Protein (6145-GH)
  • 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

AAB18278

Applications

Enzyme Activity
View all Heparinase III Proteins and Enzymes »

Product Specifications

Source

E. coli-derived p. heparinus Heparinase III protein
Gln25-Pro659, with an N-terminal Met and 6-His tag

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.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

74 kDa

SDS-PAGE

66 kDa, reducing conditions

Activity

Measured by its ability to liberate oligosaccharides from heparin.
The specific activity is >3,000 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant P. heparinus Heparinase III Protein, CF

Recombinant P. heparinus Heparinase III Protein Enzyme Activity

Recombinant P. heparinus Heparinase III Protein Enzyme Activity

Heparinase III digestion of Heparan Sulfate (200 μg) is assessed in a 5-minute kinetic assay at room temperature by monitoring absorbance at 232 nm. R&D Systems RecombinantP. heparinumHeparinase III (Catalog # 6145-GH) (orange) exhibits much greater activity than the Bacteroides Heparinase III from the competition (green).

Formulation, Preparation, and Storage

6145-GH
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: Heparinase III

Heparan sulfate is a sulfated glycosaminoglycan with the repeating disaccharide units of -4HexA1,4GlcNAc beta 1-. It is usually attached to the protein cores of proteoglycans found on the cell membrane and in the extracellular matrix where it binds to a variety of protein ligands and regulates a wide range of biological activities (1, 2). Heparan sulfate has a domain structure where sulfated regions are interspaced with less or non-sulfated regions (3, 4). Heparin shares the backbone structure with heparan sulfate but contains no non‑sulfated regions. Heparinases are a family of lyases that release unsaturated oligosaccharides from heparin and heparan sulfate upon digestion (5). Heparinase I recognizes highly sulfated regions and is more specific for heparin. Heparinase II digests both heparin and heparan sulfate. Heparinase III prefers less-sulfated regions and is more active on heparan sulfate (6, 7).

References

  1. MacArthur, J. M. et al. (2007) J. Clin. Invest. 117:153.
  2. Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
  3. Maccarana, M. et al. (1996) J. Biol. Chem. 271:17804.
  4. Linker, A. and Hovingh, P. (1975) Biochim. Biophys. Acta. 385:324.
  5. Linker, A. and Hovingh, P. (1965) J. Biol. Chem. 240:3724.
  6. Su, H. et al. (1996) Appl. Environ. Microbiol. 62:2723.
  7. Hovingh, P. and Linker, A. (1970) J. Biol. Chem. 245:6170.

Entrez Gene IDs

8254931 (P. heparinus)

Gene Symbol

PHEP_3797

UniProt

AAB18278

Additional Heparinase III Products

Product Documents for Recombinant P. heparinus Heparinase III Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant P. heparinus Heparinase III Protein, CF

For research use only

Citations for Recombinant P. heparinus Heparinase III Protein, CF

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Protocols

View specific protocols for Recombinant P. heparinus Heparinase III Protein, CF (6145-GH):

Materials
  • Assay Buffer: 50 mM Tris, 2 mM CaCl2, pH 7.5
  • Recombinant P. heparinus Heparinase III (rP. heparinus Heparinase III) (Catalog # 6145-GH)
  • Substrate: Heparin (Tocris, Catalog # 2812), 20 mg/mL stock in deionized water
  • 96 well clear UV-transparent microplate (Corning, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rP. heparinus Heparinase III to 0.5 ng/µL in Assay Buffer.
  2. Dilute Substrate to 1.5 mg/mL in Assay Buffer.
  3. Load into a plate 100 µL of the diluted rP. heparinus Heparinase III, and start the reaction by adding 200 µL of 1.5 mg/mL Substrate. Include a Substrate Blank containing 100 µL of Assay Buffer and 200 µL of 1.5 mg/mL Substrate.
  4. Read in kinetic mode for 5 minutes at an absorbance of 232 nm.
  5. Calculate specific activity:

     Specific Activity (pmoles/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 3800 M-1cm-1 
     ***Using the path correction 0.92 cm
     Note: the output of many spectrophotometers is in mOD. Per Well:

  • rP. heparinus Heparinase III: 0.05 µg
  • Substrate: 1.0 mg/mL

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