• 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.  

• Activity

  Measured by its ability to liberate oligosaccharides from heparin. The specific activity is >24,500 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Gln22-Arg384, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  42 kDa

• SDS-PAGE

  40-42 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 100 mM NaCl, 2 mM CaCl₂, pH 7.5

• Recombinant F. heparinum Heparinase I (rFhHeparinase I) (Catalog # 7897-GH)

• Substrate: Heparin (Tocris, Catalog # ), 20 mg/mL stock in deionized water

• 96-well Clear UV Plate (Costar, Catalog # 3635)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rFhHeparinase I to 0.5 µg/mL in Assay Buffer.

2. Dilute Substrate to 0.75 mg/mL in Assay Buffer.

3. Load 100 µL of diluted rFhHeparinase I into a UV plate, 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 plate in kinetic mode for 5 minutes at an absorbance of 232 nm.

5. Calculate specific activity:

     *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 Reaction:

• rFhHeparinase I: 0.05 µg

• Substrate: 0.5 mg/mL

Data Images

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. Heparinases degrade heparin and heparan sulfate glycosaminoglycans through an eliminative mechanism (3). Heparinase I from Flavobacterium   heparinum is highly active on heparin and has no activity against chondroitin sulfate and keratan sulfate (4). The enzyme readily releases highly sulfated oligosaccharides from heparin (5).

• 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. Linhardt, R. J. et al. (1986) Appl. Biochem. Biotech. 12:135.

4. Wu, Z.L. et al. (2011) Glycobiology 21:625.

5. Ernst, S. et al. (1996) Biochem. J. 315:589.

• Entrez Gene IDs:

  1073402 (B. thetaiotaomicron)

• Alternate Names:

  Heparinase I