Recombinant P. heparinus Chondroitinase AC Protein, CF

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.

• Reactivity: Ph
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant P. heparinus Chondroitinase AC Protein, CF Summary

• Additional Information: Analyzed by SEC-MALS
• Details of Functionality: Measured by its ability to hydrolyze chondroitin sulfate. The specific activity is >50,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived p. heparinus Chondroitinase AC protein
  Gln23-Lys700 with an N-terminal Met and 6-His tag
• Accession #: Q59288
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Phep_0786
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <0.01 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 78 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 66 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • 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 10^12 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant P. heparinus Chondroitinase AC Protein, CF

• Chondroitinase AC

Background

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).

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.

Bioinformatics

• Gene Symbol: Phep_0786
• Uniprot:
  • Q59288()