Collagenase from Clostridium histolyticum (C15-1198-088)

General description

Clostridium histolyticum is a pathogenic clostridium that produces collagenase. This is a mixture of enzymes containing collagenase, non-specific proteases and clostripain.

Application

Collagenase may be used:

• for the preparation of arterial tissue for the study of advanced glycosylation end products (AGE)
• for use along with other proteases for the disaggregation of human tumor, mouse kidney, human brain, lung epithelium and many other tissues.
• in liver and kidney perfusion studies, digestion of pancreas, and isolation of nonparenchymal hepatocytes
• for the preparation of viable hepatocytes from rat liver and for the isolation of fat cells from rat adipose tissue

Biochem/physiol Actions

Collagenase is activated by four gram atom calcium, per mole enzyme. It is inhibited by ethylene glycol-bis(β-aminoethyl ether) - N, N, N′,N′-tetraacetic acid, β-mercaptoethanol, glutathione, thioglycolic acid and 8-hydroxyquinoline.Collagenase enzymes and neutral protease plays an important role in the effective release of cells from tissue. Collagenase recognizes the sequence -R-Pro-8-X-Gly-Pro-R-, where X is most often a neutral amino acid.

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.

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

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.
This product also contains clostripain, nonspecific neutral protease, and tryptic activities.