Recombinant B. thetaiotaomicron Heparinase I Protein, CF

R&D Systems | Catalog # 5830-GH

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Key Product Details

  • R&D Systems E. coli-derived Recombinant B. thetaiotaomicron Heparinase I Protein (5830-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

NP_813586

Applications

Enzyme Activity
View all Heparinase I Proteins and Enzymes »

Product Specifications

Source

E. coli-derived b. thetaiotaomicron Heparinase I protein
Met1-Arg376, with Met, 6-His tag and a fusion partner at the N-terminus

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

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

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

55 kDa

SDS-PAGE

59 kDa, reducing conditions

Activity

Measured by its ability to liberate oligosaccharides from heparin.
The specific activity is >12,000 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

5830-GH
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Citrate.
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.

Background: Heparinase I

Heparin and heparan sulfate are sulfated glycosaminoglycans that share basic carbohydrate backbone structure with alternating uronic acid and N-acetylglucosamine residues (1, 2). Heparin is found in mast cells and has strong anticoagulation properties. Heparan sulfate is found on cell membrane and extracellular matrix and is involved in various biological events from cell growth, adhesion and migration to lipid metabolism. Heparin has a much higher degree of sulfation than heparan sulfate, which can be considered as a polysaccharide with regions similar to heparin interspaced with much less sulfated regions. Both heparin and heparan sulfate can be digested by heparinases, a group of bacterial lyases that are widely used as tools for processing and analyze these polysaccharides. Heparinase I from Bacteroides thetaiotaomicron (3) is a newly discovered heparinase with no activity against chondroitin sulfate and keratan sulfate (4). The enzyme readily releases tri‑sulfated and di-sulfated disaccharides from heparin and heparan sulfate.

 

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. Xu, J. et al. (2003) Science 299:2074.
  4. Luo, Y. et al. (2007) Arch. Biochem. Biophys. 460:17.

Entrez Gene IDs

1073402 (B. thetaiotaomicron)

Gene Symbol

BT_RS23555

UniProt

NP_813586

Additional Heparinase I Products

Product Documents for Recombinant B. thetaiotaomicron Heparinase I Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant B. thetaiotaomicron Heparinase I Protein, CF

For research use only

Citations for Recombinant B. thetaiotaomicron Heparinase I Protein, CF

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Protocols

View specific protocols for Recombinant B. thetaiotaomicron Heparinase I Protein, CF (5830-GH):

Materials
  • Assay Buffer: 50 mM Tris, 300 mM NaCl, 2 mM CaCl2, pH 7.5
  • Recombinant B. thetaiotaomicron Heparinase I (rBtHeparinase I) (Catalog # 5830-GH)
  • Substrate: Heparin (Tocris, Catalog # 2812), prepare a stock solution in deionized water
  • 96 well clear UV-transparent microplate (Corning, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rBtHeparinase I to 1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 0.75 mg/mL in Assay Buffer.
  3. Load into plate 100 µL of 1 ng/µL rBtHeparinase I, and start the reaction by adding 200 µL of 0.75 mg/mL Substrate. Include a Substrate Blank containing 100 µL of Assay Buffer and 200 µL of 0.75 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/M
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:

  • rBtHeparinase I: 0.1 µg
  • Substrate: 0.5 mg/mL

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