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) and for the preparation of single-cell suspensions for the establishment of primary cell culture systems. Collagenase A is recommended when yield, viability and functionality 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.

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.

Other Notes

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

Shipping conditions may vary

Preparation Note

Activator: Ca²⁺Inhibitors:Collagenase inhibitors: EDTA, EGTA, Cys, His, DTT, 2-mercapto-ethanolCollagenase is not inhibited by serum.Clostripain inhibitors: TLCKTrypsin inhibitors: aprotinin, trypsin inhibitor (egg white, soybean), serumWorking concentration: 0.5 to 2.5 mg/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.

Reconstitution

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

Specificity

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 activityCollagenase A has a balanced ratio of enzyme activities.

Unit Definition

ContentsLyophilizate, nonsterile

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).