Skip to main content
SpringerLink
Log in

pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

CD:

circular dichroism

D:

denatured state

F:

folded state

I:

intermediate state

MRE:

mean residue ellipticity

SB:

stem bromelain

References

  1. Kim, P. S., and Baldwin, R. L. (1990) Ann. Rev. Biochem., 59, 613–660.

    Google Scholar 

  2. Reyna, A. A., and Arana, A. H. (1995) Biochim. Biophys. Acta, 1248, 123–128.

    Google Scholar 

  3. Jemmings, O. A., and Wright, P. E. (1993) Science, 262, 892–895.

    Article  Google Scholar 

  4. Kuwajima, K. (1989) Proteins, 6, 87–103.

    Article  CAS  PubMed  Google Scholar 

  5. Ptitsyn, O. B. (1987) J. Protein Chem., 6, 273–293.

    Article  CAS  Google Scholar 

  6. Sanz, J. M., and Gallego, G. G. (1997) Eur. J. Biochem., 240, 328–335.

    Article  Google Scholar 

  7. Cohen, L. W., Coghlan, V. M., and Dihe, L. C. (1986) Gene, 48, 219–227.

    Article  CAS  PubMed  Google Scholar 

  8. Carne, A., and Moore, C. H. (1978) Biochem. J., 173, 73–83.

    CAS  PubMed  Google Scholar 

  9. Dubois, T., Kleinschmidt, T., Schnek, A. G., Looze, Y., and Braunitzer, G. (1988) Biol. Chem. Hoppe-Seyler, 369, 741–754.

    CAS  PubMed  Google Scholar 

  10. Topham, C. M., Salih, E., Frazao, C., Kowlessur, D., Overington, J. P., Thomas, M., Brocklehurst, S. M., Patel, M., Thomas, E. W., and Brocklehurst, K. (1991) Biochem. J., 280, 79–92.

    CAS  PubMed  Google Scholar 

  11. Seyler, H. (1989) Biol. Chem., 370, 425–434.

    Google Scholar 

  12. Ritonja, A., Rowan, A. D., Buttle, D. J., Railings, N. D., Turk, V., and Barett, A. J. (1989) FEBS Lett., 247, 419–424.

    Article  CAS  PubMed  Google Scholar 

  13. Watson, D. C., Yaguchi, M., and Lynn, K. R. (1990) Biochem. J., 266, 75–81.

    CAS  PubMed  Google Scholar 

  14. Kamphuis, I. G., Kalk, K. H., Swarte, M. B. A., and Drenth, J. (1984) J. Mol. Biol., 179, 233–257.

    Article  CAS  PubMed  Google Scholar 

  15. Baker, E. N. (1980) J. Mol. Biol., 141, 441–484.

    Article  CAS  PubMed  Google Scholar 

  16. Drenth, J., Iansonius, J. N., Koekoek, R., and Wolthers, B. G. (1971) Adv. Protein Chem., 25, 79–86.

    Article  CAS  PubMed  Google Scholar 

  17. Edwin, F., and Jagannadham, M. V. (2000) Biochim. Biophys. Acta, 1479, 69–82.

    CAS  PubMed  Google Scholar 

  18. Edwin, F., and Jagannadham, M. V. (2002) Biochem. Biophys. Res. Commun., 290, 1441–1446.

    Article  CAS  PubMed  Google Scholar 

  19. Dubey, V. K., and Jagannadham, M. V. (2003) Biochemistry, 42, 12287–12297.

    Article  CAS  PubMed  Google Scholar 

  20. Dubey, V. K., Shah, A., Jagannadham, M. V., and Kayastha, A. M. (2006) Protein Pept. Lett., 6, 545–547.

    Article  Google Scholar 

  21. Dubey, V. K., and Jagannadham, M. V. (2003) Phytochemistry, 62, 1057–1071.

    Article  PubMed  Google Scholar 

  22. Sundd, M., Kundu, S., Dubey, V. K., and Jagannadham, M. V. (2004) J. Biochem. Mol. Biol., 37, 586–596.

    CAS  PubMed  Google Scholar 

  23. Thakurta, P. G., Biswas, S., Chakrabarti, C., Sundd, M., Jagannadham, M. V., and Dattagupta, J. K. (2004) Biochemistry, 43, 1532–1540.

    Article  CAS  PubMed  Google Scholar 

  24. Patel, B. K., and Jagannadham, M. V. (2003) J. Agric. Food Chem., 51, 6326–6334.

    Article  CAS  PubMed  Google Scholar 

  25. Sundd, M., Kundu, S., and Jagannadham, M. V. (2000) J. Protein Chem., 3, 169–176.

    Article  Google Scholar 

  26. Kundu, S., Sundd, M., and Jagannadham, M. V. (1999) Biochem. Biophys. Res. Commun., 264, 635–642.

    Article  CAS  PubMed  Google Scholar 

  27. Murachi, T., and Yamazaki, M. (1970) Biochemistry, 9, 1935–1938.

    Article  CAS  PubMed  Google Scholar 

  28. Haq, S. K., Rasheedi, S., and Khan, R. H. (2002) Eur. J. Biochem., 269, 47–52.

    Article  CAS  PubMed  Google Scholar 

  29. Ahmad, B., and Khan, R. H. (2006) J. Biochem. (Tokyo), 140, 501–508.

    CAS  Google Scholar 

  30. Haq, S. K., Rasheedi, S., Sharma, P., Ahmad, B., and Khan, R. H. (2005) Int. J. Biochem. Cell Biol., 37, 361–374.

    Article  CAS  PubMed  Google Scholar 

  31. Gupta, P., Khan, R. H., and Saleemuddin, M. (2003) Arch. Biochem. Biophys., 413, 199–206.

    Article  CAS  PubMed  Google Scholar 

  32. Ahmad, B., Ansari, M. A., Sen, P., and Khan, R. H. (2006) Biopolymers, 81, 350–359.

    Article  CAS  PubMed  Google Scholar 

  33. Vanhoof, G., Cooreman, W., Lauwers, A., and Scharpe, S. (eds.) (1997) Bromelain in Pharmaceutical Enzymes, Marcel Dekker Inc., New York, pp. 131–147.

    Google Scholar 

  34. Sharpira, E., and Arnon, R. (1969) J. Biol. Chem., 244, 4989–4994.

    Google Scholar 

  35. Chen, Y. H., Yang, J. T., and Martinez, H. (1972) Biochemistry, 11, 4120–4131.

    Article  CAS  PubMed  Google Scholar 

  36. Pace, C. N. (1990) Trends Biotechnol., 8, 93–98.

    Article  CAS  PubMed  Google Scholar 

  37. Mucke, M., and Schmid, F. X. (1994) Biochemistry, 33, 12930–12935.

    Article  CAS  PubMed  Google Scholar 

  38. Soulages, J. S. (1998) Biophys. J., 75, 484–492.

    Article  CAS  PubMed  Google Scholar 

  39. Uversky, V. N., Karnoup, A. S., Seshadri, S., Doniach, S., and Fink, A. L. (1998) J. Mol. Biol., 278, 879–894.

    Article  CAS  PubMed  Google Scholar 

  40. Fink, A. L., Oberg, K. A., and Seshadri, S. (1998) Fold. Des., 3, 19–25.

    Article  CAS  PubMed  Google Scholar 

  41. Tanaka, N., Nishizawa, H., and Kunugi, S. (1997) Biochim. Biophys. Acta, 1338, 13–20.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, 202002, India

    B. Ahmad, G. M. Rathar, A. Varshney & R. H. Khan

Authors
  1. B. Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G. M. Rathar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Varshney
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. H. Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. H. Khan.

Additional information

Published in Russian in Biokhimiya, 2009, Vol. 74, No. 12, pp. 1642–1649.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM09-098, November 8, 2009.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ahmad, B., Rathar, G.M., Varshney, A. et al. pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH. Biochemistry Moscow 74, 1337–1343 (2009). https://doi.org/10.1134/S0006297909120062

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297909120062

Key words

Search

Navigation