Recombinant B. thetaiotaomicron Heparinase I Protein, CF

• Reactivity: Bthe
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant B. thetaiotaomicron Heparinase I Protein, CF Summary

• Details of Functionality: Measured by its ability to liberate oligosaccharides from heparin. The specific activity is >12,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived b. thetaiotaomicron Heparinase I protein
  Met1-Arg376, with Met, 6-His tag and a fusion partner at the N-terminus
• Accession #: NP_813586
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: BT_4675
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 55 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 59 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Citrate.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • 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 10^12 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant B. thetaiotaomicron Heparinase I Protein, CF

• Heparinase I

Background

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.

 

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.

Publications for Heparinase I (5830-GH)(1)

Publication using 5830-GH

• Wu ZL, Prather B, Ethen CM Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases. Glycobiology, 2010-12-17;21(5):625-33. 2010-12-17 [PMID: 21169395] (Enzyme Assay, Bovine, Porcine)

Bioinformatics

• Gene Symbol: BT_4675
• Uniprot:
  • NP_813586()