Collagenase from Clostridium histolyticum

Biochemical / physiological behavior.
The collagenase product is composed of a mixture of enzymes secreted by Clostridium histolyticus, and different products can be distinguished by the proportion of 10-18 compounds found in the secretase. The main components are two kinds of collagenase, Clostridium protease and a neutral protease. The synergistic action of these enzymes can degrade collagen and other intracellular substances. The role of collagenase and neutral protease is necessary for the effective release of cells from tissue. Different types of collagen are the natural substrates of collagenase. Collagenase per mole was activated by 4G atomic calcium. It was inhibited by ethylene glycol-bis (β-aminoethyl ether)-N-tetraacetic acid, β-mercaptoethanol, glutathione, mercaptoacetic acid and 8-hydroxyquinoline.

Application.
Collagenase from Clostridium histolyticus has a wide range of applications in biological research, and it can be used to prepare isolated cell suspensions. The product is suitable for the depolymerization of human tumors, mouse kidneys, human adult and fetal brains, lungs and many other epithelial tissues. It has also been proved to be effective in the study of liver and kidney perfusion, pancreatic digestion, isolation of non-parenchymal rat hepatocytes and preparation of hepatocytes. Collagenase has been used to study the preparation of arterial tissue for advanced glycation end products. The enzyme has been tested to release hepatocytes at a concentration of about 1 mg/mL. The concentration used for digestion ranges from 0.1 to 5 mg/mL.

Points for attention in preparation.
The product is equivalent to the initial 40% ammonium sulfate part Mandl, I., et al.,J. Clin.Invest., 32, 1323 (1953). The solution is usually prepared at 1-2 mg/mL in TESCA buffer (containing 50mM TES and 0.36mM calcium chloride, pH 7.4 at 37 ℃).

General description.
Collagenase is a protease that cleaves a triple helix protein called collagen. There are three types of tissue collagenases, all of which belong to the matrix metalloproteinases (MMP) family. Collagenase obtained from Clostridium histolyticus has very strong activity because it can digest collagen from both ends at temperatures as low as 4-10 ℃. The mixture of crude collagenase contains two main types of enzymes, namely collagenase and Clostridium protease.

Unit definition.
The amount of peptide released from bovine Achilles tendon collagen by a collagen digestion unit (CDU) is the same as that of 1.0 μ leucine at pH7.4,37 ℃ in the presence of calcium ions. A FALGPA hydrolysis unit hydrolyzes 1. 0 μ mole furyl acrylyl-Leu-Gly-Pro-Ala at 25 ℃ per minute. A neutral protease unit hydrolyzes casein to produce the same color as 1.0 μ mole tyrosine produced at pH7.5,37 ℃ every 5 hours. A Clostridium protease unit was hydrolyzed at pH7.6,25 ℃ in the presence of DTT at 1. 0 μ mole BAEE per minute.

Warning.
According to the information provided, the product can be stably stored for one year at-20 °C. There was no loss of FALGPA or protease activity at 37 ℃, 50 ℃ and-20 ℃ within 30 days. If the crude collagenase is sub-packaged (10mg / mL) and frozen at-20 ℃, the solution of crude collagenase can be stable. Further repeated freezing and thawing will damage the solution. The product can remain 100% active for more than 7 hours when placed on ice.