Collagenase from Clostridium histolyticum

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 reaction varies from 15 minutes to several hours and yields a satisfactory efficiency of cell dissociation without causing too much cell death. Krebs Ringer buffer with calcium and BSA is preferred and Zn²⁺ is required for activity. This enzyme is tested for suitability for the release of hepatocytes (at approx. 1 mg/mL in a total volume of 100 mL) for each rat liver.

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.

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.

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

Prepared from Type XI (C7657)