Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Zymography methods for visualizing hydrolytic enzymes

Abstract

Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Development of zymography over five decades.
Figure 2: IGZ, ISZ and IVZ readouts.
Figure 3: IGZ and transfer-gel zymography (TGZ).
Figure 4: Three different approaches for ISZ.

Similar content being viewed by others

References

  1. Gross, J. & Lapière, C.M. Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc. Natl. Acad. Sci. USA 48, 1014–1022 (1962)This was the first study of collagenolysis in tissue, which resulted in the first description of a matrix metalloproteinase (MMP-1).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Granelli-Piperno, A. & Reich, E. A study of proteases and protease-inhibitor complexes in biological fluids. J. Exp. Med. 148, 223–234 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Heussen, C. & Dowdle, E.B. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102, 196–202 (1980).

    CAS  PubMed  Google Scholar 

  4. Hibbs, M.S., Hasty, K.A., Seyer, J.M., Kang, A.H. & Mainardi, C.L. Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase. J. Biol. Chem. 260, 2493–2500 (1985). One of the first gelatin in gel zymography methods, in which a single gel system was used.

    CAS  PubMed  Google Scholar 

  5. Masure, S., Billiau, A., Van Damme, J. & Opdenakker, G. Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate. Biochim. Biophys. Acta 1054, 317–325 (1990).

    CAS  PubMed  Google Scholar 

  6. Masure, S., Proost, P., Van Damme, J. & Opdenakker, G. Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8. Eur. J. Biochem. 198, 391–398 (1991).

    CAS  PubMed  Google Scholar 

  7. Houde, M. et al. Differential regulation of gelatinase B and tissue-type plasminogen activator expression in human Bowes melanoma cells. Int. J. Cancer 53, 395–400 (1993).

    CAS  PubMed  Google Scholar 

  8. Paemen, L. et al. The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors. Biochem. Pharmacol. 52, 105–111 (1996).

    CAS  PubMed  Google Scholar 

  9. Van den Steen, P.E. et al. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9). Crit. Rev. Biochem. Mol. Biol. 37, 375–536 (2002).

    CAS  PubMed  Google Scholar 

  10. Galis, Z.S., Sukhova, G.K., Lark, M.W. & Libby, P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J. Clin. Invest. 94, 2493–2503 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Galis, Z.S., Sukhova, G.K. & Libby, P. Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue. FASEB J. 9, 974–980 (1995).

    CAS  PubMed  Google Scholar 

  12. Kaijzel, E.L., van der Pluijm, G. & Lowik, C.W. Whole-body optical imaging in animal models to assess cancer development and progression. Clin. Cancer Res. 13, 3490–3497 (2007).

    PubMed  Google Scholar 

  13. Weissleder, R., Tung, C.H., Mahmood, U. & Bogdanov, A. Jr. In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat. Biotechnol. 17, 375–378 (1999).

    CAS  PubMed  Google Scholar 

  14. Bremer, C., Tung, C.H. & Weissleder, R. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 7, 743–748 (2001).

    CAS  PubMed  Google Scholar 

  15. Crawford, B.D. & Pilgrim, D.B. Ontogeny and regulation of matrix metalloproteinase activity in the zebrafish embryo by in vitro and in vivo zymography. Dev. Biol. 286, 405–414 (2005).

    CAS  PubMed  Google Scholar 

  16. Kleiner, D.E. & Stetler-Stevenson, W.G. Quantitative zymography: detection of picogram quantities of gelatinases. Anal. Biochem. 218, 325–329 (1994).

    CAS  PubMed  Google Scholar 

  17. Kupai, K. et al. Matrix metalloproteinase activity assays: importance of zymography. J. Pharmacol. Toxicol. Methods 61, 205–209 (2010).

    CAS  PubMed  Google Scholar 

  18. Ikeda, M. et al. Inhibition of gelatinolytic activity in tumor tissues by synthetic matrix metalloproteinase inhibitor: application of film in situ zymography. Clin. Cancer Res. 6, 3290–3296 (2000).

    CAS  PubMed  Google Scholar 

  19. Wilkesman, J. & Kurz, L. Protease analysis by zymography: a review on techniques and patents. Recent Pat. Biotechnol. 3, 175–184 (2009).

    CAS  PubMed  Google Scholar 

  20. Vandooren, J. et al. Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain. World J. Biol. Chem. 2, 14–24 (2011).

    PubMed  PubMed Central  Google Scholar 

  21. McKerrow, J.H., Pino-Heiss, S., Lindquist, R. & Werb, Z. Purification and characterization of an elastinolytic proteinase secreted by cercariae of Schistosoma mansoni. J. Biol. Chem. 260, 3703–3707 (1985).

    CAS  PubMed  Google Scholar 

  22. Baragi, V.M. et al. A versatile assay for gelatinases using succinylated gelatin. Matrix Biol. 19, 267–273 (2000).

    CAS  PubMed  Google Scholar 

  23. Lê, J. et al. Quantitative zymography of matrix metalloproteinases by measuring hydroxyproline: application to gelatinases A and B. Electrophoresis 20, 2824–2829 (1999).

    PubMed  Google Scholar 

  24. Liabakk, N.B., Talbot, I., Smith, R.A., Wilkinson, K. & Balkwill, F. Matrix metalloprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9) type IV collagenases in colorectal cancer. Cancer Res. 56, 190–196 (1996).

    CAS  PubMed  Google Scholar 

  25. Curino, A. et al. Detection of plasminogen activators in oral cancer by laser capture microdissection combined with zymography. Oral Oncol. 40, 1026–1032 (2004).

    CAS  PubMed  Google Scholar 

  26. Margulies, I.M. et al. Urinary type IV collagenase: elevated levels are associated with bladder transitional cell carcinoma. Cancer Epidemiol. Biomarkers Prev. 1, 467–474 (1992).

    CAS  PubMed  Google Scholar 

  27. Li, L. et al. Inhibitory effects of GL-V9 on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-2/9. Eur. J. Pharm. Sci. 43, 393–399 (2011).

    CAS  PubMed  Google Scholar 

  28. Wang, Z. et al. Interleukin-lβ induces migration of rat arterial smooth muscle cells through a mechanism involving increased matrix metalloproteinase-2 activity. J. Surg. Res. 169, 328–336 (2011).

    CAS  PubMed  Google Scholar 

  29. Tamura, Y. et al. Profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus. J. Thromb. Thrombolysis 32, 195–200 (2011).

    PubMed  Google Scholar 

  30. Fontana, V. et al. Consistent alterations of circulating matrix metalloproteinases levels in untreated hypertensives and in spontaneously hypertensive rats: a relevant pharmacological target. Basic Clin. Pharmacol. Toxicol. 109, 130–137 (2011).

    CAS  PubMed  Google Scholar 

  31. Knier, B. et al. Effect of the plasminogen-plasmin system on hypertensive renal and cardiac damage. J. Hypertens. 29, 1602–1612 (2011).

    CAS  PubMed  Google Scholar 

  32. Todorova, L., Bjermer, L., Westergren-Thorsson, G. & Miller-Larsson, A. TGFβ-induced matrix production by bronchial fibroblasts in asthma: budesonide and formoterol effects. Respir. Med. 105, 1296–1307 (2011).

    PubMed  Google Scholar 

  33. Huang, C.Y. et al. Advanced glycation end products cause collagen II reduction by activating Janus kinase/signal transducer and activator of transcription 3 pathway in porcine chondrocytes. Rheumatology (Oxford) 50, 1379–1389 (2011).

    CAS  Google Scholar 

  34. Paemen, L., Olsson, T., Söderström, M., van Damme, J. & Opdenakker, G. Evaluation of gelatinases and IL-6 in the cerebrospinal fluid of patients with optic neuritis, multiple sclerosis and other inflammatory neurological diseases. Eur. J. Neurol. 1, 55–63 (1994).

    CAS  PubMed  Google Scholar 

  35. Rossano, R., Larocca, M. & Riccio, P. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence. J. Plant Physiol. 168, 1517–1525 (2011).

    CAS  PubMed  Google Scholar 

  36. Geib, S.M., Tien, M. & Hoover, K. Identification of proteins involved in lignocellulose degradation using in gel zymogram analysis combined with mass spectroscopy-based peptide analysis of gut proteins from larval Asian longhorned beetles, Anoplophora glabripennis. Insect Sci. 17, 253–264 (2010).

    CAS  Google Scholar 

  37. Velada, I. et al. Expression of genes encoding extracellular matrix macromolecules and metalloproteinases in avian tibial dyschondroplasia. J. Comp. Pathol. 145, 174–186 (2011).

    CAS  PubMed  Google Scholar 

  38. Métayer, S., Dacheux, F., Dacheux, J.L. & Gatti, J.L. Comparison, characterization, and identification of proteases and protease inhibitors in epididymal fluids of domestic mammals. Matrix metalloproteinases are major fluid gelatinases. Biol. Reprod. 66, 1219–1229 (2002).

    PubMed  Google Scholar 

  39. Kim, S.H., Choi, N.S. & Lee, W.Y. Fibrin zymography: a direct analysis of fibrinolytic enzymes on gels. Anal. Biochem. 263, 115–116 (1998).

    CAS  PubMed  Google Scholar 

  40. Park, S.G. et al. A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography. Proteomics 2, 206–211 (2002).

    CAS  PubMed  Google Scholar 

  41. Hasson, S.S., Theakston, R.D. & Harrison, R.A. Antibody zymography: a novel adaptation of zymography to determine the protease-neutralising potential of specific antibodies and snake antivenoms. J. Immunol. Methods 292, 131–139 (2004).

    CAS  PubMed  Google Scholar 

  42. Hughes, A.J. & Herr, A.E. Quantitative enzyme activity determination with zeptomole sensitivity by microfluidic gradient-gel zymography. Anal. Chem. 82, 3803–3811 (2010).

    CAS  PubMed  Google Scholar 

  43. Thimon, V., Belghazi, M., Labas, V., Dacheux, J.L. & Gatti, J.L. One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry. Anal. Biochem. 375, 382–384 (2008).

    CAS  PubMed  Google Scholar 

  44. Pan, D., Hill, A.P., Kashou, A., Wilson, K.A. & Tan-Wilson, A. Electrophoretic transfer protein zymography. Anal. Biochem. 411, 277–283 (2011).

    CAS  PubMed  Google Scholar 

  45. Choi, N.S. et al. Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel. Electrophoresis 30, 2234–2237 (2009).

    CAS  PubMed  Google Scholar 

  46. Choi, N.S. et al. Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel. Anal. Biochem. 386, 121–122 (2009).

    CAS  PubMed  Google Scholar 

  47. Oliver, G.W., Leferson, J.D., Stetler-Stevenson, W.G. & Kleiner, D.E. Quantitative reverse zymography: analysis of picogram amounts of metalloproteinase inhibitors using gelatinase A and B reverse zymograms. Anal. Biochem. 244, 161–166 (1997).

    CAS  PubMed  Google Scholar 

  48. Yan, S.J. & Blomme, E.A. In situ zymography: a molecular pathology technique to localize endogenous protease activity in tissue sections. Vet. Pathol. 40, 227–236 (2003).

    CAS  PubMed  Google Scholar 

  49. Duran-Vilaregut, J. et al. Role of matrix metalloproteinase-9 (MMP-9) in striatal blood-brain barrier disruption in a 3-nitropropionic acid model of Huntington's disease. Neuropathol. Appl. Neurobiol. 37, 525–537 (2011).

    CAS  PubMed  Google Scholar 

  50. Mungall, B.A. & Pollitt, C.C. In situ zymography: topographical considerations. J. Biochem. Biophys. Methods 47, 169–176 (2001).

    CAS  PubMed  Google Scholar 

  51. Hu, J., Van den Steen, P.E., Sang, Q.X. & Opdenakker, G. Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases. Nat. Rev. Drug Discov. 6, 480–498 (2007).

    CAS  PubMed  Google Scholar 

  52. Van den Steen, P.E. et al. Matrix metalloproteinases, tissue inhibitors of MMPs and TACE in experimental cerebral malaria. Lab. Invest. 86, 873–888 (2006).

    CAS  PubMed  Google Scholar 

  53. Redondo-Muñoz, J. et al. Matrix metalloproteinase-9 promotes chronic lymphocytic leukemia B cell survival through its hemopexin domain. Cancer Cell 17, 160–172 (2010).

    PubMed  Google Scholar 

  54. Agrawal, S. et al. Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis. J. Exp. Med. 203, 1007–1019 (2006). This paper illustrates the power of combining IGZ, ISZ and IHC.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Louboutin, J.P., Agrawal, L., Reyes, B.A., Van Bockstaele, E.J. & Strayer, D.S. HIV-1 gp120-induced injury to the blood-brain barrier: role of metalloproteinases 2 and 9 and relationship to oxidative stress. J. Neuropathol. Exp. Neurol. 69, 801–816 (2010).

    CAS  PubMed  Google Scholar 

  56. Oh, L.Y. et al. Matrix metalloproteinase-9/gelatinase B is required for process outgrowth by oligodendrocytes. J. Neurosci. 19, 8464–8475 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Wyatt, R.A. et al. The zebrafish embryo: a powerful model system for investigating matrix remodeling. Zebrafish 6, 347–354 (2009).

    CAS  PubMed  Google Scholar 

  58. Keow, J.Y., Herrmann, K.M. & Crawford, B.D. Differential in vivo zymography: a method for observing matrix metalloproteinase activity in the zebrafish embryo. Matrix Biol. 30, 169–177 (2011).

    CAS  PubMed  Google Scholar 

  59. Scherer, R.L., VanSaun, M.N., McIntyre, J.O. & Matrisian, L.M. Optical imaging of matrix metalloproteinase-7 activity in vivo using a proteolytic nanobeacon. Mol. Imaging 7, 118–131 (2008). An example of how nanoparticles are useful tools for IVZ with the use of near-infrared fluorophores with low tissue absorption.

    CAS  PubMed  Google Scholar 

  60. Saghatelian, A., Jessani, N., Joseph, A., Humphrey, M. & Cravatt, B.F. Activity-based probes for the proteomic profiling of metalloproteases. Proc. Natl. Acad. Sci. USA 101, 10000–10005 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Keow, J.Y., Pond, E.D., Cisar, J.S., Cravatt, B.F. & Crawford, B.D. Activity-based labeling of matrix metalloproteinases in living vertebrate embryos. PLoS ONE 7, e43434 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Smith, C. Keeping tabs on fluorescent tags. Nat. Methods 4, 755–761 (2007).

    CAS  Google Scholar 

  63. López-Otín, C. & Overall, C.M. Protease degradomics: a new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 3, 509–519 (2002). A critical review of degradomics as a methodology to study the substrate repertoires of single proteases.

    PubMed  Google Scholar 

  64. Overall, C.M. et al. Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors. Biol. Chem. 385, 493–504 (2004).

    CAS  PubMed  Google Scholar 

  65. Schilling, O. & Overall, C.M. Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites. Nat. Biotechnol. 26, 685–694 (2008).

    CAS  PubMed  Google Scholar 

  66. Dean, R.A. & Overall, C.M. Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome. Mol. Cell Proteomics 6, 611–623 (2007).

    CAS  PubMed  Google Scholar 

  67. Cauwe, B. & Opdenakker, G. Intracellular substrate cleavage: a novel dimension in the biochemistry, biology and pathology of matrix metalloproteinases. Crit. Rev. Biochem. Mol. Biol. 45, 351–423 (2010).

    CAS  PubMed  Google Scholar 

  68. Tsiatsiani, L., Gevaert, K. & Van Breusegem, F. Natural substrates of plant proteases: how can protease degradomics extend our knowledge? Physiol. Plant. 145, 28–40 (2012).

    CAS  PubMed  Google Scholar 

  69. Piccard, H. et al. “Reverse degradomics”, monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction products. Electrophoresis 30, 2366–2377 (2009). Definition of reverse degradomics as a substrate-based methodology to study the protease repertoire of single substrates. In fact, substrate zymography is a reverse degradomics method.

    CAS  PubMed  Google Scholar 

  70. Kaberdin, V.R. & McDowall, K.J. Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix. Genome Res. 13, 1961–1965 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Foltmann, B., Szecsi, P.B. & Tarasova, N.I. Detection of proteases by clotting of casein after gel electrophoresis. Anal. Biochem. 146, 353–360 (1985).

    CAS  PubMed  Google Scholar 

  72. Till, O., Baumann, E. & Linss, W. Zymography with caseogram prints: quantification of pepsinogen. Anal. Biochem. 292, 22–25 (2001).

    CAS  PubMed  Google Scholar 

  73. Kwon, M.A., Kim, H.S., Hahm, D.H. & Song, J.K. Synthesis activity-based zymography for detection of lipases and esterases. Biotechnol. Lett. 33, 741–746 (2011).

    CAS  PubMed  Google Scholar 

  74. Law, B., Hsiao, J.K., Bugge, T.H., Weissleder, R. & Tung, C.H. Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples. Anal. Biochem. 338, 151–158 (2005).

    CAS  PubMed  Google Scholar 

  75. Hattori, S., Fujisaki, H., Kiriyama, T., Yokoyama, T. & Irie, S. Real-time zymography and reverse zymography: a method for detecting activities of matrix metalloproteinases and their inhibitors using FITC-labeled collagen and casein as substrates. Anal. Biochem. 301, 27–34 (2002).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The present study was supported by funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 263307, by “Geconcerteerde OnderzoeksActies” GOA 2012 017 and GOA 2013 014, by the Fund for Scientific Research of Flanders (FWO-Vlaanderen) and by the University of Leuven Research Fund. The authors thank B. Crawford and J. Keow for providing Figure 2c.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ghislain Opdenakker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Tables 1–4 and Supplementary Note (PDF 716 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vandooren, J., Geurts, N., Martens, E. et al. Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10, 211–220 (2013). https://doi.org/10.1038/nmeth.2371

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2371

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing