Recombinant P. vulgaris Chondroitinase ABC Protein, CF

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

Recombinant P. vulgaris Chondroitinase ABC Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze chondroitin sulfate. The specific activity is >15,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived p. vulgaris Chondroitinase ABC protein
  Ala25-Pro1021, with an N-terminal Met and 6-His tag
• Accession #: P59807
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: NEWENTRY
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 113 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: 85-100 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: 100 mM Tris, 50 mM NaCl, 10 mM MgCl₂, pH 7.5
  • Recombinant P. vulgaris Chondroitinase ABC (rP. vulgaris Chondroitinase ABC) (Catalog # 6877-GH)
  • Substrate: Chondroitin sulfate (Sigma, Catalog # C6737), 10 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. vulgaris Chondroitinase ABC 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. vulgaris Chondroitinase ABC 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. vulgaris Chondroitinase ABC: 0.050 μg
  • Chondroitin sulfate: 200 μg

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.


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

Alternate Names for Recombinant P. vulgaris Chondroitinase ABC Protein, CF

• Chondroitinase ABC

Background

Chondroitinase ABC from Proteus vulgaris is a depolymerizing lyase that is especially active on various forms of chondroitin sulfate and dermatan sulfate (1, 2). Chondroitin sulfate contributes a major structural component of cartilage and provides much of the cartilage’s resistance to compression (3). Chondroitin sulfate is also found in the nervous system and at the cell surface, where it plays regulatory roles in development (4, 5) and is involved in pathways related to microbial infection (6). Dermatan sulfate is primarily located in the skin, but is also found in the blood vessels, heart valves, tendons, and lungs, where it plays roles in blood coagulation and wound healing (7). Chondroitinase ABC is also about one hundred-fold less active on hyaluronan, a nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues (8). Chondroitinase ABC can be used to study the structures and functions of these polysaccharides. For example, in one study it was found to promote axon growth following spinal cord injury, presumably through the degradation of chondroitin sulfate in close proximity to the axons (9). Chondroitinase ABC works through beta -elimination of the 1,4-hexosaminidic bond on the polysaccharides; thereby generates unsaturated disaccharides and tetrasaccharides that exhibit strong UV absorbance at 232 nm (10).

1. Oike, Y. et al. (1980) Biochem. J. 191:193.
2. Prabhakar, V. et al. (2009), J. Biol. Chem. 284:974.
3. Plaas, A.H.K et al. (1998), J. Biol. Chem. 273:12642.
4. Brittis, P.A. et al. (1992), Science 255:733.
5. Rauch, W. (1991) J. Biol. Chem. 266:14785.
6. Fried, M. and Duffy, P.E. Science 272:1502.
7. Trowbridge, JM and Gallo, RL (2002) Glycobiology 12:117R.
8. Hamai, A. et al. (1997), J. Biol. Chem. 272:9123.
9. Garcia-Alias, G. et al. (2009) Nat. Neurosci. 12:1145.
10. Huang, W. et al. (2007) J. Mol. Biol. 328:623.

Publications for Chondroitinase ABC (6877-GH)(6)

Publications using 6877-GH

• Volpi, N;Galeotti, F;Gatto, F; High-throughput glycosaminoglycan extraction and UHPLC-MS/MS quantification in human biofluids Nature protocols 2024-11-14 [PMID: 39543382] (Bioassay, Human)
• Gutierrez-Riquelme, T;Karkossa, I;Schubert, K;Liebscher, G;Packeiser, EM;Nolte, I;von Bergen, M;Murua Escobar, H;Aguilera-Rojas, M;Einspanier, R;Stein, T; Proteomic analysis of extracellular vesicles derived from canine mammary tumour cell lines identifies protein signatures specific for disease state BMC veterinary research 2024-10-26 [PMID: 39462388] (Bioassay, Canine)
• Blake, MR;Parrish, DC;Staffenson, MA;Johnson, MA;Woodward, WR;Habecker, BA; Loss of chondroitin sulfate proteoglycan sulfation allows delayed sympathetic reinnervation after cardiac ischemia-reperfusion Physiological reports 2023-05-01 [PMID: 37226390] (Western Blot, Mouse)
• KA Queisser, RA Mellema, EA Middleton, I Portier, BK Manne, F Denorme, EJ Beswick, MT Rondina, RA Campbell, AC Petrey COVID-19 generates hyaluronan fragments that directly induce endothelial barrier dysfunction JCI Insight, 2021-09-08;0(0):. 2021-09-08 [PMID: 34314391] (Bioassay, Human)
• Z Su, S Kishida, S Tsubota, K Sakamoto, D Cao, S Kiyonari, M Ohira, T Kamijo, A Narita, Y Xu, Y Takahashi, K Kadomatsu Neurocan, an extracellular chondroitin sulfate proteoglycan, stimulates neuroblastoma cells to promote malignant phenotypes Oncotarget, 2017-11-15;8(63):106296-106310. 2017-11-15 [PMID: 29290949] (Bioassay, Human)
• Adam Linder Heparin-binding protein is important for vascular leak in sepsis Intensive Care Med Exp, 2016-10-04;4(1):33. 2016-10-04 [PMID: 27704481] (Bioassay, Human)

Bioinformatics

• Gene Symbol: NEWENTRY
• Uniprot:
  • P59807()