• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

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

• Activity

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

• Source

  E. coli-derived Gln23-Lys700 with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  78 kDa

• SDS-PAGE

  66 kDa, reducing conditions

Assay Procedure

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:

   *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

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)

• Alternate Names:

  Chondroitinase AC