Recombinant P. heparinus Heparinase II Protein, CF

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Catalog # Availability Size / Price Qty
6336-GH-010
Recombinant P. heparinus Heparinase II Protein Enzyme Activity
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Product Details
Citations (1)
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Recombinant P. heparinus Heparinase II Protein, CF Summary

Product Specifications

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 >750 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived p. heparinus Heparinase II protein
Ala26-Arg772, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
86 kDa
SDS-PAGE
66-75 kDa, reducing conditions

Product Datasheets

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

6336-GH

Formulation Supplied as a 0.2 μm filtered solution in PBS.
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.

Assay Procedure

Materials
  • Assay Buffer: 100 mM Tris, pH 7.5
  • Recombinant P. heparinus Heparinase II (rPhHeparinase II) (Catalog # 6336-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 rPhHeparinase II to 20 ng/µL in Assay Buffer.
  2. Dilute Substrate to 3.0 mg/mL in Assay Buffer.
  3. Load into a plate 50 µL of the diluted rPhHeparinase II, and start the reaction by adding 50 µL of 3.0 mg/mL Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 3.0 mg/mL Substrate.
  4. Read in kinetic mode for 5 minutes at an absorbance of 232 nm.
  5. Calculate specific activity:

     Specific Activity (pmol/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 0.32 cm
     Note: the output of many spectrophotometers is in mOD Per Well:
  • rPhHeparinase II: 1.0 µg
  • Substrate: 1.5 mg/mL

Data Image

Enzyme Activity Recombinant P. heparinus Heparinase II Protein Enzyme Activity View Larger

Heparinase II digestion of Heparin Sulfate (200 μg) is assessed in a 5-minute kinetic assay by monitoring absorbance at 232 nm. R&D SystemsP. heparinusHeparinase I (catalog # 6336-GH) exhibits activity at 2595.6 pm/min/μg.

Reconstitution Calculator

Reconstitution Calculator

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Background: Heparinase II

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 cell membrane and extracellular matrix, where it binds to a variety of protein ligands and regulates a wide range of biological activities, including developmental processes, angiogenesis, blood coagulation and tumor metastasis (1, 2). Heparan sulfate has a domain structure containing sulfated regions 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 active only 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.
Alternate Names
Heparinase II

Citation for Recombinant P. heparinus Heparinase II Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.
    Authors: Wu ZL, Prather B, Ethen CM
    Glycobiology, 2011;21(5):625-33.
    Species: Bovine
    Sample Types: Protein
    Applications: Enzyme Assay

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