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C9407 Sigma-Aldrich

Collagenase from Clostridium histolyticum

powder, Suitable for the digestion and isolation of physiologically active pancreatic islet cells, suitable for cell culture

Synonym: Clostridiopeptidase A

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Description

General description

Collagenase enzyme from Clostridium histolyticum has collagen binding domain (CBD) and polycystic kidney disease-like (PKD-like) domains. It also has Ca2+ binding site for calcium binding, which is crucial for its functionality. Collagenase is used in the management of connective tissue disorder, Dupuytren′s contracture.

Application

Collagenase from Clostridium histolyticum has been used in the digestion:
• of pancreatic samples for islets isolation
• of pulmonary artery samples for pulmonary vascular smooth muscle cells (PVSMCs) isolation
• of cartilage samples for chondrocytes isolation

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.

The collagenase product is a mixture of enzymes secreted by C. histolyticum, with different products differentiated by the relative ratios of the 10-18 components found in the secreted enzymes. The main components are two collagenases, clostripain, and a neutral protease. The synergistic action of these enzymes degrade collagen and other intracellular materialThe action of both collagenase enzymes and the neutral protease is necessary for effective release of cells from tissue. Various types of collagen are the natural substrates for collagenase.

Caution

As supplied, this product is stable for one year at -20°C. There is no loss in FALGPA or protease activity in 30 days at 37°C, 50°C and -20°C. Solutions of crude collagenase are stable if frozen quickly in aliquots (at 10 mg/mL) and kept frozen at -20°C. Further freeze-thaw cycles will damage the solution. The product retains 100% activity over 7 hours when held on ice.

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.

Preparation Note

This proudct is prepared from Type XI (C7657) Collagenase from Clostridium histolyticum. It also contains clostripain, nonspecific neutral protease and tryptic activities. Solutions are typically prepared at 1-2 mg/mL in TESCA buffer (containing 50 mM TES, 0.36 mM Calcium chloride, pH 7.4 at 37°C.

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

Documents

Certificate of Analysis

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Certificate of Origin

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Pipette Controller from Brand

Cell Culture Guide
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, Cell biology, Cell culture, Diabetes, Digestions, Enzyme activity, Fermentation, Oxidations, Purification

Protocols

Enzymatic Assay of Collagenase

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

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Peer-Reviewed Papers
15

References

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

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