General description
Collagenase B is prepared from Clostridium histolyticum cultures by filtration, ammonium sulfate precipitation, dialysis, and lyophilization. Collagenase from Clostridium histolyticum is a collagenolytic enzyme. It interacts with collagen-like substances and is responsible for the degradation of collagen into small peptides. It 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) and for the preparation of single-cell suspensions for the establishment of primary cell culture systems. Collagenase B is recommended when yield and viability are important.
Application
Clostridium collagenase from Roche has been used to prepare cells from many types of tissue, such as hepatocytes, adipocytes, pancreatic islets, epithelial cells, muscle cells, endothelial cells, etc. However, suitability of each lot of the enzyme for disruption of a particular tissue should be determined empirically.
Collagenase B has been used for preparation of cells from mouse aortas and colorectal cancer tissue.
Biochem/physiol Actions
Collagenase degrades native collagen. Clostripain, trypsin-like enzymes, and neutral proteases degrade other proteins as well.
Features and Benefits
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 total proteolytic activity
Collagenase B has a normal to high collagenase activity and a higher than average clostripain activity (usually >10U/mg).
Inhibitors:
Collagenase inhibitors: EDTA, EGTA, Cys, His, DTT, 2-mercaptoethanol
Collagenase is not inhibited by serum.
Clostripain inhibitors: TLCK
Trypsin inhibitors: aprotinin, trypsin inhibitor (egg white, soybean), serum
Preparation Note
Activator: Ca2+
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.
Reconstitution in any balanced salt solution (e.g., HBSS)
Other Notes
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).
For life science research only. Not for use in diagnostic procedures.
