General description
Collagenase is a protease which cleaves the triple-helical protein called collagen. Clostridium histolyticum produces six types of collagenases namely, α, β, γ, δ, ε and λ. These collagenases are active against connective tissue and show more reactivity towards gelatin than collagen. Collagenase obtained from Clostridium histolyticum has a very strong activity, as it digests collagen from both ends, at temperatures as low as 4-10 °C.
Application
Collagenase from C. histolyticum is widely used for the: Disaggregation of many types of tissues (e.g., lung, heart, muscle, bone, adipose tissue, liver, kidney, cartilage, mammary gland, placentae, blood vessels, brain, tumors, umbilical cord)
preparation of single-cell suspensions for the establishment of primary cell culture systems. Collagenase A is recommended when yield, viability, and functionality are important.
preparation of cells from many types of tissue, such as hepatocytes, adipocytes, pancreatic islets, epithelial cells, muscle cells, endothelial cells, etc. However, the suitability of each lot of the enzyme for disruption of a particular tissue should be determined empirically.
Biochem/physiol Actions
Collagenase degrades native collagen. Clostripain, trypsin-like enzymes, and neutral proteases also degrade other proteins.
Enzyme activity:
Collagenase activity: >0.15U/mg (according to Wünsch) (+25°C, 4-phenyl-azobenzyl-oxycarbonyl-Pro-Leu-Gly-Pro-D-Arg as the substrate)
Contaminating enzyme activities: trypsin, clostripain, and neutral proteolytic activity
Collagenase A has a balanced ratio of enzyme activities.
Preparation Note
Activator: Ca2+
Inhibitors:
Collagenase inhibitors: EDTA, EGTA, Cys, His, DTT, 2-mercapto-ethanol
Collagenase is not inhibited by serum.
Clostripain inhibitors: TLCK
Trypsin inhibitors: aprotinin, trypsin inhibitor (egg white, soybean), serum
Working concentration: 0.5 to 2.5 mg/ml
Storage conditions (working solution): -15 to -25 °C
Roche recommends reconstituting only the amount of lyophilizate needed for immediate use. The reconstituted solution can be stored at -15 to -25 °C for up to one week. Avoid repeated freezing and thawing since activity decreases after reconstitution.
Keep container tightly closed in a dry and well-ventilated place
Reconstitution in any balanced salt solution (e.g., HBSS)
Other Notes
Contents
Lyophilizate, nonsterile
For life science research only. Not for use in diagnostic procedures.
Unit Definition: Collagenase from Roche is assayed in Wünsch units (1 μmol of product formed per minute at +25 °C with Wünsch substrate).
Frequently, collagenase activities are given in Mandl units (1 μmol leucine liberated from collagen in 5 hours at +37 °C).
Unfortunately, there is no consistent conversion factor between the two units of activity, since the Mandl unit depends, in part, on the concentration of contaminating proteases in the collagenase preparation, an indefinable variable. A purer collagenase preparation would actually give a lower specific activity in Mandl units than a crude preparation. Clostridium preparations typically give conversion factors of approximately 1:1800 (e.g., a particular lot of Clostridium collagenase contained approximately 0.15 Wünsch U/mg and 250 Mandl U/mg).
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