• 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 >3,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Gln25-Pro659, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  74 kDa

• SDS-PAGE

  66 kDa, reducing conditions

Assay Procedure

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 # ), 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:

     *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
  

Data Images

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)

• Alternate Names:

  Heparinase III