详细说明
- Purity>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to hydrolyze chondroitin sulfate B. The specific activity is >90,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
- SourceE. coli-derived Gln26-His506, with an N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisInconclusive results, Met predicted. Protein identity confirmed by MS analysis of tryptic fragments. - Predicted Molecular Mass55 kDa
- SDS-PAGE52-56 kDa, reducing conditions
| 6974-GH | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
| 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.
|
Assay Procedure
Materials
- Assay Buffer: 50 mM Tris, 5 mM CaCl2, pH 7.5
- Recombinant P. heparinus Chondroitin B Lyase/Chondroitinase B (rP. heparinus Chondroitinase B) (6974‑GH)
- Substrate: Chrondroitin Sulfate B (Sigma, Catalog # C3788), 25 mg/mL stock in deionized water
- 96 well UV Plate (Costar, Catalog # 3635)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rP. heparinus Chondroitinase B to 0.4 ng/μL in Assay Buffer.
- Dilute Substrate to 4 mg/mL in Assay Buffer.
- Load 50 μL of 0.4 ng/μL rP. heparinus Chondroitinase B 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.
- Read at an absorbance of 232 nm in kinetic mode for 5 minutes.
- 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 B: 0.020 μg
- Chondroitin Sulfate B: 200 μg
Background: Chondroitin B Lyase/Chondroitinase B
Chondroitinase B from P. heparinus is a depolymerizing lyase that is especially active on the sulfated polysaccharide dermatan sulfate (DS) (1, 2). DS is a sulfated polysaccharide that is abundantly located in the skin, but is also present in the blood vessels, heart valves, tendons, and lungs. DS may play roles in coagulation, cardiovascular disease, carcinogenesis, infection, wound repair, and fibrosis (3). DS with a repeating disaccharide unit of IdoA alpha 1-3GlcNAc is most closely related to chondroitin sulfate (CS) that has a repeating disaccharide unit of GlcA beta 1-3GlcNAc. Due to this similarity, DS was originally referred to as chondroitin sulfate B, and the DS-depolymerizing enzyme was named Chondroitinase B. DS contains more sulfate than CS at levels comparable to the anticoagulant drug heparin, which is the reason that purified commercial heparin is more likely to be contaminated by DS. Chondroitinase B can be used to specifically degrade and distinguish DS in glycosaminoglycan preparations (4, 5, 6).
- References:
- Oike, Y. et al. (1980) Biochem. J. 191:193.
- Prabhakar, V. et al. (2009) J. Biol. Chem. 284:974.
- Trowbridge, J.M. and Gallo, R.L. (2002) Glycobiology 12:117R.
- Gu, K. et al. (1995) Biochem. J. 312:356.
- Tkalec, A.L. et al. (2000) Appl. Environ. Microbiol. 66:29.
- Wu, Z.L. et al. (2011) Glycobiology 21:625.
- Entrez Gene IDs:8251878 (P. heparinus)
- Alternate Names:Chondroitin B Lyase; Chondroitinase B
400-6226-992