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C7657

Sigma-Aldrich

Collagenase from Clostridium histolyticum

Type XI, 2-5 FALGPA units/mg solid, ≥800 CDU/mg solid

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Synonym(s):
Clostridiopeptidase A
CAS Number:
Enzyme Commission number:
EC Number:
MDL number:
NACRES:
NA.54

biological source

Clostridium histolyticum

Quality Level

form

powder

specific activity

≥800 CDU/mg solid
2-5 FALGPA units/mg solid

mol wt

68-130 kDa

solubility

TESCA buffer (50 mM TES, 0.36 mM Calcium chloride, pH 7.4): soluble 0.05-0.1 mg/mL at 37 °C

application(s)

diagnostic assay manufacturing

storage temp.

−20°C

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Application

Clostridiopeptidase A has 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.

Biochem/physiol Actions

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.
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.

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.

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.

Pictograms

Health hazard

Signal Word

Danger

Hazard Statements

Precautionary Statements

Hazard Classifications

Resp. Sens. 1

Storage Class Code

11 - Combustible Solids

WGK

WGK 1

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Personal Protective Equipment

dust mask type N95 (US), Eyeshields, Gloves

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Protocols

To measure collagenase activity, N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala is used in a continuous spectrophotometric rate determination at 345 nm. Collagenase hydrolyzes collagen peptide bonds.

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