Application

Collagenase from Clostridium histolyticum has been used: to harvest osteosarcoma 143B cells from collagen scaffoldin tissue digestion of islets and carotid arteryin the digestion of chorionic villiin enzymatic dissociation for ATP-based tumour chemosensitivity assay (ATP-TCA) in solid tumors

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.

Clostridium histolyticum produces two classes of collagenases. Clostridial collagenases are found to be highly effective than mammalian collagenases. It is capable of cleaving at multiple cleavage sites within the collagen triple helix. It is implicated in the bacterial invasion in gas gangrene.

Effective release of cells from tissue requires the action of both 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 is used for digestion. The duration of reaction varyies 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 conc., in a total volume of 100 mL for each rat liver.

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.