Skip to main content
SpringerLink
Log in

pH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

Acid unfolding pathway of conalbumin (CA), a monomeric glycoprotein from hen egg white, has been investigated using far- and near-UV CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe 1-anilino-8-napthalene sulfonate (ANS) and dynamic light scattering (DLS). We observe pH-dependent changes in secondary and tertiary structure of CA. It has native-like α-helical secondary structure at pH 4.0 but loss structure at pH 3.0. The CA existed exclusively as a pre-molten globule state and molten globule state in solution at pH 4.0 and pH 3.0, respectively. The effect of pH on the conformation and thermostability of CA points toward its heat resistance at neutral pH. DLS results show that MG state existed as compact form in aqueous solutions with hydrodynamic radii of 4.7 nm. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

ANS:

1-anilino-8-napthalene sulfonate

CA:

Conalbumin

DLS:

Dynamic light scattering

GnHCl:

Guanidine hydrochloride

MG:

Molten globule

MRE:

Mean residue ellipticity

P d :

Polydispersity

T m :

Mid-point temperature

References

  1. Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., & Razgulyaev, O. I. (1990). Evidence for a molten globule state as a general intermediate in protein folding. FEBS Letters, 262, 20–24.

    Article  PubMed  CAS  Google Scholar 

  2. Arai, M., Ito, K., Inobe, T., Nakao, M., Maki, K., Kamagata, K., et al. (2002). Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering. Journal of Molecular Biology, 321, 121–132.

    Article  PubMed  CAS  Google Scholar 

  3. Hsieh, H. C., Kumar, T. K., Chiu, C. C., & Yu, C. (2005). Equilibrium unfolding of an oligomeric protein involves formation of a multimeric intermediate state(s). Biochemical and Biophysical Research Communications, 326, 108–114.

    Article  PubMed  CAS  Google Scholar 

  4. Sridevi, K., Lakshmikanth, G. S., Krishnamoorthy, G., & Udgaonkar, J. B. (2004). Increasing stability reduces conformational heterogeneity in a protein folding intermediate ensemble. Journal of Molecular Biology, 337, 699–711.

    Article  PubMed  CAS  Google Scholar 

  5. Nishimura, C., Dyson, H. J., & Wright, P. E. (2008). The kinetic and equilibrium molten globule intermediates of apoleghemoglobin differ in structure. Journal of Molecular Biology, 378, 715–725.

    Article  PubMed  CAS  Google Scholar 

  6. Paci, E., Greene, L. H., Jones, R. M., & Smith, L. J. (2005). Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach. FEBS Journal, 272, 4826–4838.

    Article  PubMed  CAS  Google Scholar 

  7. Chen, B. L., Baase, W. A., Nicholson, H., & Schellman, J. A. (1992). Folding kinetics of T4 lysozyme and nine mutants at 12°C. Biochemistry, 31, 1464–1476.

    Article  PubMed  CAS  Google Scholar 

  8. Nakao, M., Maki, K., Arai, M., Koshiba, T., Nitta, K., & Kuwajima, K. (2005). Characterization of kinetic folding intermediates of recombinant canine milk lysozyme by stopped-flow circular dichroism. Biochemistry, 44, 6685–6692.

    Article  PubMed  CAS  Google Scholar 

  9. de Sousa Rde, C., Coimbra, J. S., da Silva, L. H., da Silva, Mdo. C., Rojas, E. E., & Vicente, A. A. (2009). Thermodynamic studies of partitioning behavior of lysozyme and conalbumin in aqueous two-phase systems. Journal of Chromatography B, 877, 2579–2584.

    Article  Google Scholar 

  10. Stevens, L. (1996). Egg proteins: What are their functions? Science Progress, 79, 65–87.

    PubMed  CAS  Google Scholar 

  11. Ko, K. Y., Mendonca, A. F., & Ahn, D. U. (2008). Influence of zinc, sodium bicarbonate, and citric acid on the antibacterial activity of ovotransferrin against Escherichia coli O157:H7 and Listeria monocytogenes in model systems and Ham. Poultry Science, 87, 2660–2670.

    Article  PubMed  CAS  Google Scholar 

  12. Stevens, L. (1991). Egg white proteins. Comparative Biochemistry and Physiology B, 100, 1–9.

    Article  CAS  Google Scholar 

  13. Kelly, S. M., & Price, N. C. (2000). The use of circular dichroism in the investigation of protein structure and function. Current Protein & Peptide Science, 1, 349–384.

    Article  CAS  Google Scholar 

  14. Woodworth, R. C., & Schade, A. L. (1959). Conalbumin: a rapid, high-yield preparation from egg white. Archives of Biochemistry and Biophysics, 82, 78–82.

    Article  PubMed  CAS  Google Scholar 

  15. Chen, Y. H., Yang, J. T., & Martinez, H. M. (1972). Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry, 11, 4120–4131.

    Article  PubMed  CAS  Google Scholar 

  16. Pace, C. N., & Scholtz, M. (1997). Protein Structure, A Practical Approach. In T. E. Creighton (Ed.), Measuring the conformational stability of a protein (p. 383). New York: Oxford University Press.

    Google Scholar 

  17. Ahmad, E., Rahman, S. K., Khan, J. M., Varshney, A., & Khan, R. H. (2010). Phytolacca americana lectin (Pa-2; pokeweed mitogen): An intrinsically unordered protein and its conversion into partial order at low pH. Bioscience Reports, 30, 125–134.

    Article  CAS  Google Scholar 

  18. Larsson, T., Wedborg, M., & Turner, D. (2007). Correction of inner-filter effect in fluorescence excitation-emission matrix spectrometry using Raman scatter. Analytica Chimica Acta, 2, 357–363.

    Article  Google Scholar 

  19. Venyaminov, S. Y., & Yang, J. T. (1996). In G. D. Fasman (Ed.), Circular dichroism and the conformational analysis of biomolecules (pp. 69–109). New York: Plenum Press.

    Google Scholar 

  20. Kelly, S. M., & Price, N. C. (1997). The application of circular dichroism to studies of protein folding and unfolding. Biochimica et Biophysica Acta, 1338, 161–185.

    Article  PubMed  CAS  Google Scholar 

  21. Varshney, A., Ahmad, B., Rabbani, G., Kumar, V., Yadav, S., & Khan, R. H. (2010). Acid-induced unfolding of didecameric keyhole limpet hemocyanin: detection and characterizations of decameric and tetrameric intermediate states. Amino Acids, 39, 899–910.

    Article  PubMed  CAS  Google Scholar 

  22. Ahmad, E., Fatima, S., Khan, M. M., & Khan, R. H. (2010). More stable structure of wheat germ lipase at low pH than its native state. Biochimie, 92, 885–893.

    Article  PubMed  CAS  Google Scholar 

  23. Rossi, M., & Schiraldi, A. (1992). Thermal denaturation and aggregation of egg proteins. Thermochimica Acta, 199, 115–123.

    Article  CAS  Google Scholar 

  24. Eftink, M. R., & Ghiron, C. A. (1987). Does the fluorescence quencher acrylamide bind to proteins? Biochimica et Biophysica Acta, 3, 343–349.

    Google Scholar 

  25. Hanakam, F., Gerisch, G., Lotz, S., Alt, T., & Seelig, A. (1996). Binding of hisactophilin I and II to lipid membranes is controlled by a pH-dependent myristoyl-histidine switch. Biochemistry, 35, 11036–11044.

    Article  PubMed  CAS  Google Scholar 

  26. Grimsley, G. R., Shaw, K. L., Fee, L. R., Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L., et al. (1999). Increasing protein stability by altering long-range coulombic interactions. Protein Science, 8, 1843–1849.

    Article  PubMed  CAS  Google Scholar 

  27. Fink, A. L., Calciano, L. J., Goto, Y., Kurotsu, T., & Palleros, D. R. (1994). Classification of acid denaturation of proteins: Intermediates and unfolded states. Biochemistry, 33, 12504–12511.

    Article  PubMed  CAS  Google Scholar 

  28. Pace, C. N., Alston, R. W., & Shaw, K. L. (2000). Charge-charge interactions influence the denatured state ensemble and contribute to protein stability. Protein Science, 9, 1395–1398.

    Article  PubMed  CAS  Google Scholar 

  29. Sheshadri, S., Lingaraju, G. M., & Varadarajan, R. (1999). Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large delta Cp’s. Protein Science, 8, 1689–1695.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Council of Scientific and Industrial Research (CSIR) funded project New Delhi, India grant No. 37(1456)/10/EMR-II. G. Rabbani acknowledged CSIR, New Delhi, India for financial assistance in the form of Senior Research Fellow (SRF). A. H. Khan, University of Glasgow, Scotland is greatly acknowledged for the editing and valuable suggestions during the preparation of this manuscript.

Author information

Authors and Affiliations

  1. Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, 202 002, India

    Gulam Rabbani, Ejaz Ahmad, Nida Zaidi & Rizwan Hasan Khan

Authors
  1. Gulam Rabbani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ejaz Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nida Zaidi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rizwan Hasan Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rizwan Hasan Khan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rabbani, G., Ahmad, E., Zaidi, N. et al. pH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White. Cell Biochem Biophys 61, 551–560 (2011). https://doi.org/10.1007/s12013-011-9237-x

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-011-9237-x

Keywords

Search

Navigation