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  • Bacteroides Heparinase I

    Description

    Bacteroides Heparinase I cloned from Bacteroides eggerthii, also called Heparin Lyase I, is active on heparin and the highly sulfated domains of heparan sulfate. The reaction yields oligosaccharide products containing unsaturated uronic acids which can be detected by UV spectroscopy at 232 nm.

    In contrast to the Flavobacterium heparinum Heparinase I which cleaves the glycosidic bond between N-sulfated hexosamines and 2-O-sulfated iduronic acid residues, the Bacteroides Heparinase I cleaves between these same residues as well as the 2-O-sulfated glucuronic acid residues. The 2-O-sulfated uronic acid residue is essential for the activity of Bacteroides Heparinase I and 6-O-sulfation of GlcNS does not hinder enzyme activity. While Bacteroides Heparinase I cleaves 2-O sulfated iduronic acid and 2-O sulfated glucuronic acid residues at similar rates, the Flavobacterium heparinum Heparinase I has a much higher rate of cleavage for 2-O sulfated iduronic acid residues (1). Limited digest of porcine mucosal heparin with Flavobacterium heparinum Heparinase I results in sulfated heparin oligosaccharides structures previously reported (2). Limited digest of porcine mucosal heparin with the Bacteroides Heparinase I results in heparin oligosaccharides with a lower extent of sulfation as reported (3).



    Product Source

    Cloned from Bacteroides Eggerthii and expressed in pET21A.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Bacteroides Heparinase Reaction Buffer (10X)10X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will liberate 1.0 μmol unsaturated oligosaccharides from porcine mucosal heparin per minute at 30°C and pH 7.0 in a total reaction volume of 100 μl.

    Unit Definition Assay
    Two fold dilutions of Bacteroides Heparinase I are incubated with 1 mg/ml porcine mucosal heparin substrate in 1X Bacteroides Heparinase Reaction Buffer, in a 100 μl reaction. The reaction mix is incubated at 30°C. Liberation of unsaturated oligosaccharides is detected by real-time UV spectroscopy at 232 nm.

    Reaction Conditions

    1X Bacteroides Heparinase Reaction Buffer
    Incubate at 30°C

    1X Bacteroides Heparinase Reaction Buffer:
    20 mM Tris-HCl
    100 mM NaCl
    1.5 mM CaCl2
    pH 7 @ 25°C

    Storage Temperature

    -80°C

    Storage Conditions

    20 mM Tris-HCl
    100 mM NaCl
    5 mM CaCl2
    1 mM Na2EDTA
    pH 7.5 @ 25°C

    Heat Inactivation

    100°C for 1 min

    Molecular Weight

    Apparent: 42 kDa

    Quality Control

    Quality Assurance Statement

    • No contaminating exoglycosidase, sulfatase, uronidase or proteolytic activity could be detected (ND).

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Glycosidase Activity (TLC):
      The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide.  The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
    • Protease Activity (SDS-PAGE):

      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.

    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    Notes

    1. Avoid repeated freeze-thaw cycles.
    2. Reaction Conditions:
      1. Combine 10 μl of 1 mg/ml heparin substrate, 10 μl Bacteroides Heparinase Reaction Buffer and H2O in a total reaction volume of 100 μl. 
      2. Add 1 μl Bacteroides Heparinase I. 
      3. Incubate reaction at 30°C for 1–24 hours (monitor absorbance at 232 nm for determination of partial or complete digestion). 
      Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.

    References

    1. Landry, D. et al New England Biolabs, Inc.. Unpublished observation
    2. Yamada, S., Murakami, T., Tsuda, H., Yoshida K., Sugahara, K. (1995). J. Biol. Chem. 270, 8696-8705.
    3. Merchant, Z.M., Kim, Y.S., Rice, K.G., Linhardt, R. (1985). J. Biol. Chem. 229, 369-377.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What are Bacteroides Heparinase Enzymes?
    2. What is the difference between Bacteroides Heparinase I, II and III?
    3. Can Bacteroides Heparinase I, II, and III be used together in one digest?
    4. What is the optimal pH range for Bacteroides Heparinase I?
    5. Is Bacteroides Heparinase I activated by Calcium?

    Bacteroides Heparinase I cleaves highly sulfated heparin/HS chains

    Bacteroides Heparinase I is most active between pH 6.5-7.5

    Bacteroides Heparinase I is calcium dependent and has greatest activity in the presence of 1.5 - 5mM CaCl2.

    Bacteroides Heparinase I can be used in double digests with Bacteroides Heparinase II and III using the Heparinase reaction buffer.