Assessing collagenase enzyme activities, what do the results mean?

A common question often asked in selecting a collagenase containing enzyme product for use in a cell isolation protocol is what value does the enzyme activity analysis on a Certificate of Analysis provide? This is difficult to answer when using crude or enriched collagenase products since the assay results may not reflect the effectiveness of the enzyme to degrade native collagen. This brief overview correlates collagenase biochemistry to their analysis in different enzymatic assays and emphasizes the advantages of using purified, well characterized collagenase enzymes to improve the reproducibility of cell isolation procedures.

Numerous studies using purified TDEs have shown that purified collagenase and neutral protease are required for successful isolation of hepatocytes¹, islets², bone cells³, and endothelial cells⁴. Typically, class I (C1) & class 2 (C2) collagenase from C. histolyticum and neutral protease(s) are required to degrade the extracellular matrix (ECM) proteins found in mammalian tissues. The ECM is a macromolecular complex that binds cells to the tissue. Collagen is the predominant protein found in the ECM and histochemical studies analyzing the affect of TDEs on minced rat pancreatic tissue indicate that the critical protein that holds the ECM together is collagen^5,6.

This observation is consistent with the recommendation by many experts that collagenase is a critical enzyme for successful cell isolation^7-9. Since collagenase, by definition, is the only enzyme(s) that degrades the triple helical domain that is found in all forms of native collagen, then what information do we need to properly interpret assays that assess C. histolyticum collagenase activity?

C. histolyticum synthesizes two classes of collagenase, class I (C1) and class II (C2) that have molecular weights of 116 kDa (C1_116kDa) and 114 kDa (C2_114kDa), respectively¹⁰. Each enzyme has four protein domains: a catalytic domain at the amino terminal end followed by a linking domain(s) and collagen binding domain(s). C1_116kDa has a catalytic domain, one linking domain and two collagen binding domains whereas C2_114kDa has a catalytic domain, two linking domains and one collagen binding domain. Studies have shown that only collagenase with a catalytic domain and at least one collagen binding domain  can degrade native collagen. The function of the collagen binding domain binds to collagen’s triple helical domain  Once bound, the catalytic domain can cut the helix leading to unraveled (i.e., denatured) collagen that can be further degraded by neutral protease or other molecular forms of collagenase. This means that there are only 3 forms of enzyme that are active in degradation of native collagen: C1_116kDa, C1_100kDa, and C2_114kDa. The C1_100kDa is a degraded form of C1 that contains one collagen binding domain and is often found in crude, enriched, or purified preparations of C. histolyticum collagenase.  

The assays commonly used to assess collagenase enzyme activity are listed below. Two important points should be noted. The Wunsch or FALGPA assays are commonly used by many manufacturers to assess collagenase activity. These assays are precise but biased since they primarily detect C2 activity. Furthermore, these assays assess the activity of the catalytic domain so the results cannot distinguish between enzymes with or without a collagen binding domain. The collagenase activity detected by collagen degradation assays has an absolute requirement for the presence of a collagen binding domain, For many years, the Mandl assay has been used to assess this activity but recently, VitaCyte has developed a fluorescent microplate assay that uses FITC labeled collagen fibrils,substrate¹¹. This substrate is resistant to trypsin and preferentially detects C1_116kDa : the specific CDA of C1_116kDa is about 10x higher than the C1_100kDa or C2_114kDa forms of collagenase.

 If you are having trouble finding the appropriate lot of collagenase for your cell isolation procedure or want to avoid the non-productive activity required for qualifying new lots of crude or enriched collagenase, contact the cell isolation experts at VitaCyte. VitaCyte offers 3 different purified collagenase products that are defined by the proportion of C1_116kDa and their specific CDA activity. CIzyme Collagenase HA (high CDA activity) has the highest proportion of this form of the enzyme (specific CDA of > 30,000 CDA U/mg protein). Decreasing proportions of C1_116kDa are found in Collagenase MA (moderate activity) and LA (low activity) products where the specific CDAs are 15,000-30,000 CDA U/mg and 7,000-15,000 CDA U/mg, respectively. Product selection is dependent on the application. Recent results indicate that a high proportion of C1_116kDa (Collagenase HA) is critical for successful human islet isolation¹⁶ whereas the other forms of collagenase have been successfully used in hepatocyte and adipose stem cell isolation.

Each collagenase product is highly purified, consistently manufactured and rigorously characterized for specific Wunsch and CDA activity. Once the appropriate concentration of the collagenase and neutral protease have been established for your cell isolation procedure, the need to pre-qualify lots of product is minimized since the dissociation enzyme requirements are defined. These products are stable on storage and can tolerate several freeze-thaw cycles.

Now there is a real choice for your application which can be reliably used even if there are long periods of time between isolation experiments. For more specific product information please refer to our product guide.  

 Reference List

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2.   Linetsky E., Bottino R., Lehmann R., Alejandro R., Inverardi L., and Ricordi C. (1997) Improved human islet isolation using a new enzyme blend, liberase. Diabetes 46, 1120

3.   Hefley T.J. (1987) Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone. J Bone and Mineral Research 2, 505-16.

4.   Suggs W., Van Wart H., and Sharefkin J.B. (1992) Enzymatic harvesting of adult human saphenous vein endothelial cells: use of a chemically defined combination of two purified enzymes to attain viable cell yields equal to those attained by crude bacterial collagenase preparations. Journal of Vascular Surgery 15, 205-13.

5.   Wolters G.H., Vos-Scheperkeuter G.H., van Deijnen J.H., and van Schilfgaarde R. (1992) An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation. Diabetologia. 35, 735-42.

6.   Vosscheperkeuter G.H., Vansuylichem P.T.R., Vonk M.W.A., Wolters G.H.J., and Vanschilfgaarde R. (1997) Histochemical Analysis of the Role of Class I and Class Ii Clostridium Histolyticum Collagenase in the Degradation of Rat Pancreatic Extracellular Matrix for Islet Isolation. Cell Transplantation 6, 403-12.

7.   Kin T., Johnson P.R.V., Shapiro A.M.J., and Lakey J.R.T. (2007) Factors influencing the collagenase digestions phase of human islet isolation. Transplantation  83, 7-12.

8.   Berry M.N., Grivell A.R., Grivell M.B., and Phillips J.W. (1997) Isolated hepatocytes–past, present and future. Cell Biol Toxicol 13, 223-33.

9.   Williams S.K., McKenney S., and Jarrell B.E. (1995) Collagenase lot selection and purification for adipose tissue digestion. Cell Transplantation. 4[3], 281-9.

10.  Matsushita O. and Okabe A. (2001) Clostridial hydrolytic enzymes degrading extracellular components. Toxicon 39, 1769-80.

11.  McCarthy R.C., Spurlin B., Wright M.J., Breite A.G., Sturdevant L.K., Dwulet C.S., and Dwulet F.E. (2008) Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation. Transplantation Proceedings 40, 339-42. 2008.

12.  Wünsch E. and Heidrich H.-G. (1963) Zur quantitativen bestimmung der kollagenase. Hoppe-Seyler’s Zeitschrift Physiologische Chemie 333, 149-51.

13.  Van Wart H.E. and Steinbrink D.R. (1981) A continuous spectrophotometric assay for Clostridium histolyticum collagenase. Analytical Biochemistry 113, 356-65. 1981.

14   Mandl I., Keller S., and Manahan J. (1964) Multiplicity of Clostridium histolytcum collagenases. Biochemistry 3, 1737-41.

15   Peterkofsky B. (1982) Bacterial Collagenase. Methods in Enzymology 82, 453-71.

16.  Balamurugan A.N., Breite A.G., Anazawa T., Loganathan G., Wilhelm J.J., Papas K.K., Dwulet F.E., McCarthy R.C., and Hering B.J. (2010) Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplantation 89, 954-61.