Enzymes for Cell Detachment and Tissue Dissociation

BioFiles 2006, 1.2, 3.

BioFiles 2006, 1.2, 3.

Collagenase

Collagenase cleaves the peptide bonds in native, triple-helical collagen. Because of its unique ability to hydrolyze native collagen, it is widely used in isolation of cells from animal tissue. Collagenases occur in a variety of microorganisms and many different animal cells.1 The most potent collagenase is the “crude” collagenase secreted by the anaerobic bacterium Clostridium histolyticum. C. histolyticum collagenases have molecular weights from 68,000 to 125,000 Da and are metalloproteinases that require zinc and calcium.

The original 1953 fermentation and purification process described by MacLennan, Mandl, and Howes2 was first adopted by Sigma- Aldrich and eventually improved upon for higher activity products. “Crude” collagenase refers to the fact that the material is actually a mixture of several different enzymes in addition to collagenase that act together to break down tissue. It is now known that two forms of the collagenase enzyme are present.3,4 With a few exceptions different commercial collagenases are all made from C. histolyticum, or are recombinant versions where E. coli expresses a gene cloned from C. histolyticum. We provide a lot reservation service. You may purchase small quantities from one or more lots and reserve larger quantities until your evaluation is complete.

The different collagenase products in the tables were developed by Sigma-Aldrich because they digested different types of tissue (muscle, pancreas, heart, adipose) better than others. Besides meeting enzyme activity specifications, every lot of many Sigma- Aldrich collagenase products must pass digestion tests with various tissues from rats. Products that are also described as “cell culture tested” have undergone additional testing with mammalian cell lines to verify that they are not cytotoxic.

Sterile-filtered (0.2 μm) versions prepared from some of the more popular collagenase products are also available.

Sigma-Aldrich’s purified collagenase products have only trace amounts of caseinase (proteolytic) or clostripain activities. The purified Type VII Collagenase is also offered in cell culture tested and sterile-filtered versions.

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Enzymatic Assays for Collagenase

The isoforms I and II of purified collagenase differ in their specificities and relative activities on native collagen and synthetic substrates. These two collagenases can be distinguished by their preference for one of the two different substrates used in Sigma- Aldrich’s assays. The Collagenase Digestive Unit (CDU) assay10,11 measures predominantly the Collagenase I activity, which cleaves two of the three helical chains in the long, undenatured collagen protein. Collagenase II activity is measured by this enzyme’s ability to cut a short synthetic peptide, N-[3-(2-Furyl)acryloyl)]-Leu-Gly- Pro-Ala (FALGPA, see Cat. No. F5135), in a second collagenase digestive assay.12,13 Purified preparations of either isoform I or II have been shown to be less effective at digesting various types of collagen or mammalian tissue when compared to a mixture of both forms of the enzyme. Obviously the combination of true collagenase and the different native proteases, clostripain and aminopeptidases that have evolved in nature assist each other in digesting the collagen in different animal tissues. For tissue digestions the crude collagenase products have always been the most effective. Some researchers have tried mixtures of chromatographically purified collagenase with a protease such as trypsin or subtilisin to digest tissue.

In addition to the CDU and FALGPA assays for collagenase activities Sigma-Aldrich tests each product lot for caseinase,14,15 clostripain and tryptic activities to evaluate the proteolytic activities in our collagenase products. The caseinase assay is the most important of the three for measuring the proteolytic activity that assists the digestion of animal tissue. Because the clostripain present in crude collagenase must be reduced (e.g., by treatment with dithiothreitol) in order to be active this enzyme probably contributes little to the tissue dissociation process in the laboratory. It is monitored because some researchers have reported that clostripain may be damaging or toxic.

Many collagenase products that meet enzymatic specifications are also application-tested with various tissues obtained from rats. Type II (C6885, C1764) and Type VIII (C2139) collagenase lots are tested for the ability to release adipose (fat) cells from rat epididymal fat pads.5 Fat cells are then screened for metabolic activity by measuring glucose oxidation rates with and without insulin addition. Type IV (C5138, C1889) and Type VIII (C2139) lots have been tested for the ability to release viable cells from rat liver.7 Type V (C9263, C2014), Type XI (C7657, C4785, C9407, and C9697) and Type S (C6079) collagenase lots must release intact islets of Langerhans from rat pancreas to pass their product test.8

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Materials

     

References

  1. Harper, E., Collagenases, Annu. Review of Biochemistry, 49, 106 (1980).
  2. MacLennan, J. D., et al., J. Clin. Invest. 32, 1317 (1953)
  3. Bond, M.D., and Van Wart, H.E., Biochemistry, 23, 3085 (1984)
  4. Matsushita, O., et al., J. Bacteriology, 181, 923 (1999)
  5. Rodbell, M., J. Biol. Chem., 239, 375 (1964)
  6. Fain, J.N., Meth. Enzymol., 35, 555 (1975)
  7. Seglen, P.O., Methods in Cell Biology, 13, 29 (1976)
  8. Lacy, P.E., and Kostianovsky, M., Diabetes, 16, 35 (1967)
  9. Buitrago, A., et al., Biochem. Biophys. Res. Commun., 79, 823 (1977)
  10. Moore S., and Stein, W.H., J. Biol. Chem., 176, 367 (1948)
  11. Sigma-Aldrich quality control test procedure
  12. Van Wart, H.E., and Steinbrink, D.R., Anal. Biochem., 113, 356 (1981)
  13. Sigma-Aldrich quality control test procedure
  14. Anson, M.L., J. Gen. Physiol., 22, 79 (1938)
  15. Sigma-Aldrich quality control test procedure

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