Advertisement

Zymography pp 115-121 | Cite as

A Sensitive, Rapid, and Specific Technique for the Detection of Collagenase Using Zymography

  • Shivcharan Prasad
  • Ipsita RoyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1626)

Abstract

In-gel zymography is a commonly employed tool to identify active enzymes in a quantitative and qualitative manner. In this work, apart from the incorporation of substrate which is traditionally employed in zymography, the identification of collagenase by incubation of the enzyme resolved on a polyacrylamide gel with substrate solution is described. The two methods are quite fast and result in specific detection of bacterial collagenase.

Key words

Collagen Collagenase Matrix metalloproteinase Zymography 

Notes

Acknowledgements

Partial financial support from Department of Science and Technology is acknowledged. The authors thank K. Thanzami for her contribution to the work described here.

References

  1. 1.
    Vandenbroucke RE, Libert C (2014) Is there new hope for therapeutic matrix metalloproteinase inhibition? Nat Rev Drug Discov 13:904–927CrossRefPubMedGoogle Scholar
  2. 2.
    Lombard C, Saulnier J, Wallach J (2005) Assays of matrix metalloproteinases (MMPs) activities: a review. Biochimie 87:265–272CrossRefPubMedGoogle Scholar
  3. 3.
    Birkedal-Hansen H, Taylor RE (1982) Detergent-activation of latent collagenase and resolution of its component molecules. Biochem Biophys Res Commun 107:1173–1178CrossRefPubMedGoogle Scholar
  4. 4.
    Snoek-van Beurden PA, Von den Hoff JW (2005) Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques 38:73–83CrossRefPubMedGoogle Scholar
  5. 5.
    Thanzami K, Roy I (2008) A sensitive, rapid and specific technique for the detection of collagenase using zymography. Electrophoresis 29:1585–1588CrossRefPubMedGoogle Scholar
  6. 6.
    Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10:211–220CrossRefPubMedGoogle Scholar
  7. 7.
    Gross J, Lapière CM (1962) Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc Natl Acad Sci U S A 48:1014–1022CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Harrington DJ (1996) Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun 64:1885–1891PubMedPubMedCentralGoogle Scholar
  9. 9.
    Matsushita O, Koide T, Kobayashi R, Nagata K, Okabe A (2001) Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase. J Biol Chem 276:8761–8770CrossRefPubMedGoogle Scholar
  10. 10.
    Yoshihara K, Matsushita O, Minami J, Okabe A (1994) Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase. J Bacteriol 176:6489–6496CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Duarte AS, Correia A, Esteves AC (2014) Bacterial collagenases – a review. Crit Rev Microbiol. doi: 10.3109/1040841X.2014.904270 PubMedGoogle Scholar
  12. 12.
    Moore S, Stein WH (1948) Photometric ninhydrin method for use in the chromatography of amino acids. J Biol Chem 176:367–388PubMedGoogle Scholar
  13. 13.
    Mandl I, MacLennan J, Howes E (1953) Isolation and characterization of proteinase and collagenase from Cl. histolyticum. J Clin Investig 32:1323–1329CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Walker JM (2009) SDS polyacrylamide gel electrophoresis of proteins. In: Walker JM (ed) The protein protocols handbook. Humana Press, Totowa, NJ, pp 177–185CrossRefGoogle Scholar
  15. 15.
    Leber TM, Balkwill FR (1997) Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels. Anal Biochem 249:24–28CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of BiotechnologyNational Institute of Pharmaceutical Education and ResearchS.A.S. NagarIndia

Personalised recommendations