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) and for the preparation of single cell suspensions for the establishment of primary cell culture systems. Collagenase D is recommended when functionality and integrity of cell-surface proteins are important.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.

Features and Benefits

ContentsLyophilizate, nonsterile

General description

Collagenase D is prepared from Clostridium histolyticum cultures by filtration, ammonium sulfate precipitation, dialysis, and lyophilization.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Physical form

Lyophilizate, nonsterile.

Preparation Note

Activator: Ca²⁺Working concentration: 0.5 - 2.5mg/mlStorage conditions (working solution): -15 to -25°CRoche 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.Inhibitors:Collagenase inhibitors: EDTA, EGTA, Cys, His, DTT, 2-mercaptoethanolCollagenase is not inhibited by serum.Clostripain inhibitors: TLCKTrypsin inhibitors: aprotinin, trypsin inhibitor (egg white, soybean)Enzyme activity:Collagenase activity: >0.15 U/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 activityCollagenase D has a normal to high collagenase activity and very low tryptic activity (usually ﹤0.2 units/mg, BAEE as substrate).

Reconstitution

Reconstitution in any balanced salt solution (e.g., HBSS)

Specificity

Collagenase degrades native collagen. Clostripain, trypsin-like enzymes, and neutral proteases degrade other proteins as well.

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).