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Recombinant P. heparinus Chondroitinase AC Protein, CF

R&D Systems | Catalog # 8384-GH

Analyzed by SEC-MALS
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Key Product Details

  • R&D Systems E. coli-derived Recombinant P. heparinus Chondroitinase AC Protein (8384-GH)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

Q59288

Applications

Enzyme Activity
View all Chondroitinase AC Proteins and Enzymes »

Product Specifications

Source

E. coli-derived p. heparinus Chondroitinase AC protein
Gln23-Lys700 with an N-terminal Met and 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.01 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

78 kDa

SDS-PAGE

66 kDa, reducing conditions

Activity

Measured by its ability to hydrolyze chondroitin sulfate.
The specific activity is >50,000 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant P. heparinus Chondroitinase AC Protein, CF

Recombinant P. heparinus Chondroitinase AC Protein SEC-MALS.

Recombinant P. heparinus Chondroitinase AC Protein (Catalog # 8384-GH) has a molecular weight (MW) of 77-85 kDa as analyzed by SEC-MALS, suggesting that this protein is a monomer.

Formulation, Preparation, and Storage

8384-GH
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
Shipping The product is shipped with polar packs. 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, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: Chondroitinase AC

Chondroitinase AC from P. heparinum is a depolymerizing lyase that degrades chondroitin sulfates A and C, but not chondroitin sulfate B (1). Both chondroitin sulfate A and C contain D-glucuronic acid, N-acetylgalactosamine, and sulfate residues in equal molar quantities (2). The sulfate ester is at the 4-O position of N-acetylgalactosamine residues on chondroitin sulfate A and at the 6-O position of N-acetylgalactosamine residues on chondroitin sulfate C. In contrast chondroitin sulfate B contains L-iduronic acid instead of glucuronic acid (3). Chondroitinase AC cleaves, via an elimination mechanism, chondroitin sulfate chains between N-acetylgalactosamine and glucuronic acid residues. The reaction yields oligosaccharide products, primarily disaccharides, containing unsaturated uronic acids that can be detected by UV spectroscopy. The enzyme shows approximately equal activity with chondroitin sulfates A orC (3). Chondrotinase AC can be used to specifically degrade chondroitin A and C and distinguish among different species of glycosaminoglycans (4).

References

  1. Tkalec, A.L. et al. (2000) Appl. Environ. Microbiol. 66:29.
  2. Saito, H., et al. (1968) J. Biol. Chem. 243:1536.
  3. Gu, K. et al. (1995) Biochem. J. 312:569.
  4. Wu, Z.L. et al. (2011) Glycobiology 21:625.

Entrez Gene IDs

8251875 (P. heparinus)

Gene Symbol

PHEP_0786

UniProt

Q59288

Additional Chondroitinase AC Products

Product Documents for Recombinant P. heparinus Chondroitinase AC Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant P. heparinus Chondroitinase AC Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant P. heparinus Chondroitinase AC Protein, CF (8384-GH):

Materials
  • Assay Buffer: 25 mM Tris, 50 mM NaCl, 10 mM MgCl2, pH 7.5
  • Recombinant P. heparinus Chondroitinase AC (rP. heparinus Chondroitinase AC) (Catalog # 8384-GH)
  • Substrate: Chondroitin Sulfate (Sigma, Catalog # C6737), 50 mg/mL stock in deionized water
  • 96 well UV Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rP. heparinus Chondroitinase AC to 1 ng/μL in Assay Buffer.
  2. Dilute Substrate to 4 mg/mL in Assay Buffer.
  3. Load 50 μL of 1 ng/μL rP. heparinus Chondroitinase AC into the plate, and start the reaction by adding 50 μL of 4 mg/mL Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 4 mg/mL Substrate.
  4. Read at an absorbance of 232 nm in kinetic mode for 5 minutes.
  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:
  • rP. heparinus Chondroitinase AC: 0.050 μg
  • Chondroitin Sulfate: 200 μg

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