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Enzyme Explorer

Collagenase Guide

 General Information about Sigma-Aldrich Collagenase Products

Collagenases, enzymes that break down the native collagen that holds animal tissues together, are made by a variety of microorganisms and by many different animal cells1. The most potent collagenase is the "crude" collagenase secreted by the anaerobic bacteria Clostridium histolyticum. 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 besides collagenase that act together to break down tissue. It is now known that two forms of the collagenase enzyme are present3, 4. With a few exceptions different commercial collagenase are all made from C. histolyticum, or are recombinant versions where Escherichia coli expresses a gene cloned from C. histolyticum.

 

 Description of Sigma-Aldrich's Collagenase Products

The different Collagenase products in the tables below were developed by Sigma-Aldrich because they digested some 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. Some 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 mm) versions prepared from some of the more popular collagenase products are also listed below.

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.
 

Crude Collagenase for General Use
 

Product Comments FALGPAa CDUsb
C0130 For general use 0.25 – 1.0 > 125
C1639, Type I-S Sterile-filtered version of C0130 0.25 – 1.0 > 125
C9891, Type IA Second version of Type I for general use 0.5 – 2.0 > 125
C2674, Type IA Cell culture tested version of C9891 0.5 – 2.0 > 125
C5894, Type IA-S Sterile-filtered version of C9891 0.5 – 2.0 > 125
C9722, Type IA-S Cell culture tested and sterile-filtered version of C9891 0.5 – 2.0 > 125

a, b FALGPA and Collagenase Digestive Units (CDUs) both given as Units per mg/solid

 

Crude Collagenase, Use-Tested
 

Product Comments FALGPAa CDUsb
C6885, Type II Tested as suitable for digestion and release of fat (adipose) cells with undamaged cell-surface receptors for insulin5, 6 0.5 – 2.0 > 125
C1764, Type II-S Sterile-filtered version of C6885 0.5 – 2.0 > 125
C5138, Type IV Tested as suitable for digestion and release of viable rat liver cells (hepatocytes)7 0.5 – 2.0 > 125
C1889, Type IV-S Sterile-filtered version of C5138 0.5 – 2.0 > 125
C2139, Type VIII Passed both the adipose tissue test (see C6885)5, 6 and the hepatic tissue test (see C5138)7 0.5 – 2.0 > 125
C9263, Type V Tested as suitable for digestion and release of islet of Langerhans (insulin-producing cells) from rat pancreas8, 9 1.0 – 3.0 > 125
C2014, Type V-S Sterile-filtered version of C9263 1.0 – 3.0 > 125
C7657, Type XI Has highest collagenase activity - tested as suitable for digestion and release of islet of Langerhans (insulin-producing cells) from rat pancreas8, 9 2.0 – 5.0 > 1,200
C9407, Type XI Cell culture tested version of C7657 2.0 – 5.0 > 1,200
C4785, Type XI-S Sterile-filtered version of C7657 2.0 – 5.0 > 1,200
C9697, Type XI-S Cell culture tested and sterile-filtered version of C7657 2.0 – 5.0 > 1,200

a, b FALGPA and Collagenase Digestive Units (CDUs) both given as Units per mg/solid

 

Chromatographically Purified Collagenase
 

Product Comments FALGPAa CDUsb
C0255, Type III Purified collagenase that has trace amounts of protease activity 4 – 12 ≥ 250 
C0773, Type VII Purified collagenase substantially free of protease and Clostripain 4 – 12 1,000 - 3,000
C2799, Type VII Cell culture tested version of C0773 4 – 12 1,000 - 3,000
C2399, Type VII-S Sterile-filtered version of C0773 4 – 12 1,000 - 3,000
C9572, Type VII-S Cell culture tested and sterile-filtered version of C0773 4 – 12 1,000 - 3,000

a, b FALGPA and Collagenase Digestive Units (CDUs) both given as Units per mg/solid

 

Crude Collagenase with Proteolytic Activity Inhibitor
 

Product Comments FALGPAa CDUsb
C6079, Type S Blend of Type XI Collagenase with tryptic protease inhibitor. Tested as suitable for digestion and release of islet of Langerhans (insulin-producing cells) from rat pancreas8, 9 2 – 5 Minimum of 1,000

a, b FALGPA and Collagenase Digestive Units (CDUs) both given as Units per mg/solid

 

Sigma Collagenase Blends™

These products were developed to allow more lot-to-lot reproducibility in collagenase digestions. The proportion of purified collagenase (Blend F) to purified clostridial neutral protease is varied in Blends H and L to provide researchers with a range of digestion options.
 

Product Comments FALGPAa Proteasec
C7926, Sigma Blend F Predominantly true collagenases. Because FALGPA activity is high compared to CDU it is assumed that the Type II form of collagenase is mostly present Min. 2.0 < 10
C8051, Sigma Blend H Some neutral protease activity included with the collagenase activity Min. 1.0 Min. 10
C8176, Sigma Blend L More neutral protease activity is included with true collagenase < 1.0 Min. 40

a, c FALGPA and Collagenase Digestive Units (CDUs) both given as Units per mg/solid

 

 Collagenase Assays

The Type I and Type II forms of the purified collagenase enzymes differ in their specificities and relative activities on native collagen and synthetic substrates. These two collagenases can be mostly 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 Product No. F5135), in a second collagenase digestive assay12, 13. Purified preparations of either Collagenase 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 this enzyme. A purified collagenase containing only the Collagenase I and II forms of this enzyme is less effective at digesting tissue than the whole crude collagenase or combinations of the purified collagenase and various proteases. 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 Caseinase14, 15, Clostripain and Tryptic activities to look at the proteolytic enzymatic activities in the 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 use-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 pads5. 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 liver7. 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 test8.

 

 Factors that Affect Tissue Digestion-Dissociation by Collagenase

Based on our own R&D and from discussions with customers it is clear that the way a particular tissue is dissected and prepared has a significant effect on the speed and efficiency of any tissue digestion-dissociation with collagenase. Differences in the ages of the tissue donors can also be a major source of variation over time. Make sure that calcium ions are present in the digestion buffers at 5 mM. Chelating agents EGTA and EDTA can severely inhibit Collagenase activity by removing Calcium ions required for enzyme stability and activity. β-mercaptoethanol16, cysteine16 and 8-hydroxyquinoline-5-sulfonate16 are other inhibiting substances. A new lot of Collagenase with higher specific activity could cause excessive cell death at an established concentration. In that case use less collagenase and/or add BSA or serum (up to 0.5% and 5-10% respectively) to stabilize the cells to further digestion.
 

Problem Cause Solution Ref.
Digestion is poor Inactive Enzymes

Inadequate Ca++


Insufficient Enzymes
Store Collagenase cold and dry.

Store collagenase solution in frozen aliquots.
Include 5 mM Ca++ in collagenase solution.

Use more collagenase. Try Sigma Blends with more protease activity.
6


7
Released Cells are Trapped DNA released from broken cells

Inadequate Non-protein gums
Reduce agitation
Add DNAse**

Flush tissue well before digesting it***
Use no Mg++ in digestion solution
Add hyaluronidase with enzymes**
7
9

7
7
18
Cells Are Killed Excess protease


pH changes


Too little oxygen
Reduce exposure to proteases*
Add albumin or heated serum

Use buffers (e.g. HEPES) instead of HCO-3.
Check and readjust pH often

Aerate digestion solution (air or O2)
Digest faster by using more enzymes




7


19
Cell Yield is Low Enzyme balance


Adhesion factors
Use more Collagenase*
Include elastase with Collagenase**

Perfuse tissue first to remove Ca++***

20

7, 22
Many Cells are Damaged Excessive Protease


Physical damage
Use less protease*
Add albumin or heated serum

Handle tissue and cells very gently
21


7, 22
New Lot Doesn’t work Lot variation Use fractionated Sigma collagenase “blends” (Sigma Product No. C7926, C8051 or C8176)*  

* The separately prepared Collagenase and protease enzymes in the “Sigma Blend” products (No. C7926, C8051 or C8176) give reproducible control of how much of each is used.

** DNAse will be inactivated by the shear of excessive stirring, and added enzymes may be digested by the neutral protease present in the Collagenase.

*** Use EGTA (or EDTA) to remove Ca++ and flush away microorganisms, then wash tissue with buffer to remove the chelating agent. Do not add EGTA or EDTA to the enzyme solutions!

 

References

  1. Harper, E., Collagenases, Annu. Review of Biochemistry 49, 1063 (1980).
  2. MacLennan, J. D., Mandl, I., and Howes, E. L., Bacterial Digestion of Collagen, J. Clin. Invest. 32, 1317 (1953)
  3. Bond, M.D., and Van Wart, H.E. , Characterization of the Individual Collagenases from Clostridium histolyticum, Biochemistry 23, 3085 (1984)
  4. Matsushita, O., Jung, C.-M., Katayama, S., Minami, J., Takahashi, Y., and Okabe, A., Gene duplication and Multiplicity of Collagenases in Clostridium histolyticum. J of Bacteriology 181, 923 (1999)
  5. Rodbell, M., Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis, J. Biol. Chemistry 239, 375 (1964)
  6. Fain, J.N., Isolation of Free Brown and White Fat Cells, Methods in Enzymology 35, 555 (1975)
  7. Seglen, P.O., Preparation of Isolated Rat Liver Cells, Methods in Cell Biology 13, 29 (1976)
  8. Lacy, P.E., and Kostianovsky, M., Method for the Isolation of Intact Islets of Langerhans from the Rat Pancreas, Diabetes 16, 35 (1967)
  9. Buitrago, A., Gylfe, E., Henriksson, C., Pertoft, H., Rapid Isolation of Pancreatic Islets from Collagenase Digested Pancreas by Sedimentation Through Percol at Unit Gravity, Biochem. Biophys. Res. Commun. 79, 823 (1977)
  10. Moore S., and Stein, W.H., Photometric Ninhydrin Method, J. Biol. Chemistry 176, 367 (1948)
  11. “Enzymatic Assay of Collagenase. Collagen Digestion Assay”, Sigma-Aldrich quality control test procedure.
  12. Van Wart, H.E., and Steinbrink, D.R., A Continuous Spectrophotometric Assay for Clostridium histolyticum Collagenase, Anal. Biochem. 113, 356 (1981)
  13. Enzymatic Assay of Collagenase Using FALGPA as the Substrate”, Sigma-Aldrich quality control test procedure.
  14. Anson, M.L., The Estimitation of Pepsin, Trypsin, Papain and Cathepsin with Hemoglobin. J. Gen. Physiol. 22, 79 (1938)
  15. Enzymatic Assay of Caseinase (Collagenase Products)”, Sigma-Aldrich quality control test procedure.
  16. Seifter, S., Gallop, P.M., Klein, L., and Meilman, E., Studies on Collagen, Part II. Properties of Purified Collagenase and Its Inhibition. J. Biol. Chem. 234:285 (1959)
  17. Enzyme Handbook, D.Schomberg and M. Salzmann, Editors. Springer-Verlang, 1991, Vol. 5. 18. Berry, M.N., and Friend, D.S., High-yield Preparation of Isolated Rat-liver Parenchymal Cells, J. Cell Biol. 43, 506 (1969)
  18. Bellemann, P., Gebhardt, R., and Mecke, D., An Improved Method for the Isolation of Hepatocytes from Liver Slices. Selective Removal of Trypan Blue-dyeable Cells, Anal. Biochem. 81, 408 (1977)
  19. Ives, H.E., Schultz G.S., Galardy R.E. and Jamieson J.D., Preparation of Functional Smooth Muscle Cells from the Rabbit Aorta. J. Expt. Med. 148, 1400 (1978)
  20. Fain, J.N. and Loken, S.C., Response of Trypsin-treated Brown and White Fat Cells to Hormones. Preferential Inhibition of Insulin Action. J. Biol. Chem. 244, 3500 (1969)
  21. Berry, M.N., Edwards, A.M., and Barritt, G.J., Isolated Hepatocytes; Preparation, Properties and Applications. Elsevier. 1991.