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C7657 Sigma

Collagenase from Clostridium histolyticum

release of physiologically active rat pancreatic islets tested, Type XI, 2-5 FALGPA units/mg solid, ≥800 CDU/mg solid

Synonym: Clostridiopeptidase A

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Description

Biochem/physiol Actions

Collagenase is activated by four gram atom calcium per mole enzyme. It is inhibited by ethylene glycol-bis(beta-aminoethyl ether) - N, N, N′,N′-tetraacetic acid, beta-mercaptoethanol, glutathione, thioglycolic acid and 8-hydroxyquinoline.

Effective release of cells from tissue requires the action of collagenase enzymes and the neutral protease. Collagenase is activated by four gram atom calcium (Ca2+) per mole enzyme. The culture filtrate is thought to contain at least 7 different proteases ranging in molecular weight from 68-130 kDa. The pH optimum is 6.3-8.8. The enzyme is typically used to digest the connective components in tissue samples to liberate individual cells. Collagenase treatment can cause some cells to die. Typically, concentrations varying from 0.1 to 5 mg/mL are used for digestion. The duration of the reaction varies from 15 minutes to several hours to yield a satisfactory efficiency of cell dissociation without causing too much cell death. Krebs Ringer buffer with calcium and BSA is preffered and Zn2+ is required for activity. This enzyme is tested for the release of hepatocytes at approximately 1 mg/mL concentration, in a total volume of 100 mL for each rat liver.

Preparation Note

A stock solution may be prepared by dissolving 0.05-0.1 mg/mL of collagenase in 50 mM TES buffer containing 0.36 mM calcium chloride (TESCA), pH 7.4, at 37 °C.

Quality

Also contains clostripain, nonspecific neutral protease, and tryptic activities.

Unit Definition

One collagen digestion unit (CDU) liberates peptides from collagen from bovine achilles tendon equivalent in ninhydrin color to 1.0 μmole of leucine in 5 hours at pH 7.4 at 37 °C in the presence of calcium ions. One FALGPA hydrolysis unit hydrolyzes 1.0 μmole of furylacryloyl-Leu-Gly-Pro-Ala per min at 25°C. One Neutral Protease unit hydrolyzes casein to produce color equivalent to 1.0 μmole of tyrosine per 5 hr at pH 7.5 at 37°C. One Clostripain Unit hydrolyzes 1.0 μmole of BAEE per min at pH 7.6 at 25°C in the presence of DTT.

Application

Clostridiopeptidase A has been used to study the avoidance of the progression of necrotic tissue in neonates as well as to evaluate the effect of clostridiopeptidase A collagenase (CAC) in a rat model of postoperative peritoneal adhesion. Clostridiopeptidase A has also been used in a study to describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen.

This product has been used in the isolation of pancreatic ductal adenocarcinoma (PDAC) cell lines and the tissue culture of pancreatic fibroblasts.

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Safety & Documentation

Safety Information

Symbol 
Signal word 
Danger
Hazard statements 
Precautionary statements 
Personal Protective Equipment 
RIDADR 
NONH for all modes of transport
WGK Germany 
1
Protocols & Articles

Articles

Enzymes for Cell Detachment and Tissue Dissociation

Collagenase cleaves the peptide bonds in native, triple-helical collagen. Because of its unique ability to hydrolyze native collagen, it is widely used in isolation of cells from animal tissue. Colla...
BioFiles 2006, 1.2, 3.
Keywords: Anaerobic, Biochemistry, Biofiles, Cell biology, Cell culture, Diabetes, Digestions, Enzyme activity, Fermentation, Methods, Oxidations, Purification, Type

Protocols

Enzymatic Assay of Collagenase

The continuous spectrophotometric rate determination (A345, Light path = 1 cm) is based on the following reaction:
Keywords: Extinction coefficient

Related Content

Enzymes & Proteins

Application Index | Enzyme Index | Substrate Index | Inhibitor Index | Cofactor Index | Lectin Index
Keywords: Cell signaling, Diagnostic, Drug discovery, Molecular biology

Peer-Reviewed Papers
15

References

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substrate

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F5135 N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala
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