Current Science

Open Access Open Access  Restricted Access Subscription Access

Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes


Affiliations
1 Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata 700 0032, India
 

Inactivation of an enzyme as it begins to unfold, along with the conformational perturbations which follow, can provide an insight into dynamics of the unfolding pathway. Urea gradient electrophoresis combined with zymography is a sensitive technique which provides a continuous visual profile of a proteolytic enzyme undergoing denaturation and inactivation simultaneously. Trypsin has been used as a reference protease to validate and standardize the method by correlating inactivation profile generated in zymography with a solution state assay. Stem bromelain, a cysteine endopeptidase was used as a case study to evaluate this methodology. The method highlighted the effect rendered by the substrate on the stability of the proteolytic domain of the enzyme, as it undergoes urea-induced unfolding. Transverse urea gradient zymography combined with molecular modelling of stem bromelain, where the disulphide bonds have been reduced, indicated that the evolutionary retention of Cys23-Cys63 could be attributed to localized stabilization imparted by this bond to the catalytic site. This method encompasses various dimensions to extend the understanding of structure-function relationship in denaturant-induced unfolding pathways of proteases.

Keywords

Protein Unfolding, Stem Bromelain, Urea Gradient, Zymography.
User
Notifications
Font Size

  • Creighton, T. E., Electrophoretic analysis of the unfolding of proteins by urea. J. Mol. Biol., 1979, 129, 235–264.

  • Gentile, F., Veneziani, B. M. and Sellitto, C., Polyacrylamide gel electrophoresis in discontinuous transverse urea-gradient gels. Anal. Biochem., 1997, 244, 228–232.

  • Goldenberg, D. P. and Creighton, T. E., Gel electrophoresis in studies of protein conformation and folding. Anal. Biochem., 1984, 138, 1–18.

  • Goldenberg, D. P., Analysis of protein conformation by gel electrophoresis. In Protein Structure: A Practical Approach (ed. Creighton, T. E.), IRL Press, Oxford, UK, 1989, pp. 225–250.

  • Creighton, T. E., Kinetic study of protein unfolding and refolding using urea gradient electrophoresis. J. Mol. Biol., 1979, 137, 61–80.

  • Vandooren, J., Geurts, N., Martens, E., Van den Steen, P. E. and Opdenakker, G., Zymography methods for visualizing hydrolytic enzymes. Nat. Methods, 2013, 10, 211–220.

  • Gorg, A., Postel, W. and Johann. P., pH, urea and substrate gradients for the optimization of ultrathin polyacrylamide gel zymograms. J. Biochem. Biophys. Methods, 1985, 10, 341–350.

  • Tyagi, S. C., Lewis, K., Pikes, D., Marcello, A., Majumdar, V. S., Smiley, L. M. and Moore, C. K., Stretch-induced membrane type matrix metalloproteinase and tissue plasminogen activator in cardiac fibroblast cells. J. Cell Physiol., 1998, 176, 374–382.

  • Brumano, M. H. and Oliveira, M. G., Urea-induced denaturation of beta-trypsin: an evidence for a molten globule state. Protein Pept. Lett., 2004, 11, 133–140.

  • Delaage, M. and Lazdunski, M., Trypsinogen, trypsin, trypsinsubstrate and trypsin-inhibitor complexes in urea solutions. Eur. J. Biochem., 1968, 4, 378–384.

  • Lopez-Garcia, B., Hernandez, M. and Segundo, B. S., Bromelain, a cysteine protease from pineapple (Ananas comosus) stem, is an inhibitor of fungal plant pathogens. Lett. Appl. Microbiol., 2012, 55, 62–67.

  • Cohen, L. W., Coghlan, V. M. and Dihel, L. C., Cloning and sequencing of papain-encoding cDNA. Gene, 1986, 48, 21–27.

  • Chakrabarti, C., Biswas, S., Kundu, S., Sundd, M., Jagannadham, M. V. and Dattagupta, J. K., Crystallization and preliminary X-ray analysis of ervatamin B and C, two thiol proteases from Ervatamia coronaria. Acta. Crystallogr. D. Biol. Crystallogr., 1999, 55, 1074–1075.

  • Carne, A. and Moore, C. H., The amino acid sequence of the tryptic peptides from actinidin, a proteolytic enzyme from the fruit of Actinidia chinensis. Biochem. J., 1978, 173, 73–83.

  • Kamphuis, I. G., Kalk, K. H., Swarte, M. B. A. and Drenth, J., Structure of papain refined at 1.65 Å resolution. J. Mol. Biol. 1984, 179, 233–256.

  • Trivedi, M. V., The role of thiols and disulphids in protein chemical and physical stability. Curr. Protein Pept. Sci., 2009, 10, 614–625.

  • Sarath, G., Motte, R. S. D. L. and Wagner, F. W., Protease assay methods. In Proteolytic Enzymes: A Practical Approach (eds Beynon, R. J. and Bond, J. S.), IRL Press, Oxford University, Oxford, 1996, pp. 25–55.

  • Murachi, T. and Yamazaki, M., Changes in conformation and enzymic activity of stem bromelain at alkaline media. Biochemistry, 1970, 9, 1935–1938.

  • Kaino, S., Furui, T., Hatano, S., Kaino, M., Okita, K. and Nakamura. K., Two-dimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice. Electrophoresis, 1998, 19, 782–787.

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

  • Rowan, A. D. and Buttle, D. J., Pineapple cysteine endopeptidases. Methods Enzymol., 1994, 244, 555–568.

  • Albalasmeh, A. A., Berhe, A. A. and Ghezzehei, T. A., A new method for rapid determination of carbohydrate and total carbon concentrations using UV spectrophotometry. Carbohydr. Polym., 2013, 97, 253–261.

  • Sasaki, M., Takeda, S., Kato, T. and Matsuba, K., Antigenicities of stem bromelain. Contribution of three-dimensional structure and individual amino acid residues. J. Biochem., 1980, 87, 817–824.

  • Keil, B., Trypsin. In The Enzymes III Hydrolysis: Peptide Bonds (ed. Boyer, P. D.), Academic Press, London, 1971, pp. 250–273.

  • Uversky, V. N., Cracking the folding code. Why do some proteins adopt partially folded conformations, whereas other don’t? FEBS Lett., 2002, 514, 181–183.

  • Yasuda, Y., Takahashi, N. and Murachi, T., The composition and structure of carbohydrate moiety of stem bromelain. Biochemistry, 1970, 9, 25–32.

  • Ishihara, H., Takahashi, N., Oguri, S. and Tejima, S., Complete structure of the carbohydrate moiety of stem bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides. J. Biol. Chem., 1979, 254, 10715–10719.

  • Creighton, T. E., Protein folding. Biochem. J., 1990, 270, 1–16.

  • Ahmad, B., Shamim, T. A., Haq, S. K. and Khan, R. H., Identification and characterization of functional intermediates of stem bromelain during urea and guanidine hydrochloride unfolding. J. Biochem., 2007, 141, 251–259.


Abstract Views: 392

PDF Views: 111




  • Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes

Abstract Views: 392  |  PDF Views: 111

Authors

Kanika Sharma
Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata 700 0032, India
Debasish Bhattacharyya
Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata 700 0032, India

Abstract


Inactivation of an enzyme as it begins to unfold, along with the conformational perturbations which follow, can provide an insight into dynamics of the unfolding pathway. Urea gradient electrophoresis combined with zymography is a sensitive technique which provides a continuous visual profile of a proteolytic enzyme undergoing denaturation and inactivation simultaneously. Trypsin has been used as a reference protease to validate and standardize the method by correlating inactivation profile generated in zymography with a solution state assay. Stem bromelain, a cysteine endopeptidase was used as a case study to evaluate this methodology. The method highlighted the effect rendered by the substrate on the stability of the proteolytic domain of the enzyme, as it undergoes urea-induced unfolding. Transverse urea gradient zymography combined with molecular modelling of stem bromelain, where the disulphide bonds have been reduced, indicated that the evolutionary retention of Cys23-Cys63 could be attributed to localized stabilization imparted by this bond to the catalytic site. This method encompasses various dimensions to extend the understanding of structure-function relationship in denaturant-induced unfolding pathways of proteases.

Keywords


Protein Unfolding, Stem Bromelain, Urea Gradient, Zymography.

References





DOI: https://doi.org/10.18520/cs%2Fv111%2Fi8%2F1340-1348