Advertisement

Biochemistry (Moscow)

, Volume 80, Issue 4, pp 463–472 | Cite as

Denaturation properties and folding transition states of leghemoglobin and other heme proteins

  • Pijush Basak
  • Niloy Kundu
  • Rudradip Pattanayak
  • Maitree BhattacharyyaEmail author
Article

Abstract

This work reports unfolding transitions of monomeric heme proteins leghemoglobin (Lb), myoglobin (Mb), and cytochrome c (Cyt c) utilizing UV-Vis spectra, steady-state and time-resolved fluorescence methods. Conformational stabilities of the native “folded” state of the proteins and their “unfolded” states were investigated in the light of a two-state transition model. Two-state transition values for ΔGD (298K) were obtained by denaturation with the chaotropic agents urea and guanidium hydrochloride (GdnHCl). The free energy value of Lb is the lowest compared to Cyt c and Mb along the denaturation pathway. The m value is also the lowest for Lb compared to Cyt c and Mb. The m value (a measure of dependence of ΔGD on denaturant concentration) for Cyt c and Mb is lower when it is denatured with urea compared to GdnHCl. The UV-Vis absorbance maximum and steady state fluorescence emission maximum were drastically red shifted in the presence of a certain denaturant concentration both in cases of Mb and Lb, but the scenario is different for Cyt c. The results are analyzed using a two-state transition model. The lifetime data clearly indicate the presence of an intermediate state during denaturation. The unfolding transition can modulate the conformation, stability, and surface exposure of these biologically important proteins.

Key words

chaotropic agent denaturation heme protein Gibbs free energy two-state transition 

Abbreviations

Cyt c

cytochrome c

GdnHCl

guanidium hydrochloride

Hb

hemoglobin

Lb

leghemoglobin

Mb

myoglobin

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Monera, O. D., Kay, C. M., and Hodges, R. S. (1994) Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions, Protein Sci., 3, 1984–1991.CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Amiri, M., Jnakeje, K., and Albani, J. R. (2010) Origin of fluorescence lifetimes in human serum albumin. Studies on native and denatured protein, J. Fluoresc., 20, 651–656.CrossRefPubMedGoogle Scholar
  3. 3.
    Appleby, C. A. (1969) Properties of leghemoglobin in vivo and its isolation as ferrous oxyleghemoglobin, Biochim. Biophys. Acta, 188, 222–226.CrossRefPubMedGoogle Scholar
  4. 4.
    Aviram, I., and Weissmann, C. (1978) Spectrophotometric and fluorometric study of the denaturation of Euglena cytochrome c552, Biochemistry, 10, 2020–2025.CrossRefGoogle Scholar
  5. 5.
    Schechter, A. N., and Epstein, C. J. (1968) Spectral studies on the denaturation of myoglobin, J. Mol. Biol., 35, 567–589.CrossRefGoogle Scholar
  6. 6.
    Otting, G., Liepinsh, E., and Wuthrich, K. (1991) Protein hydration in aqueous solution, Science, 254, 974–980.CrossRefPubMedGoogle Scholar
  7. 7.
    Denisov, V. P., and Halle, B. (1996) Protein hydration dynamics in aqueous solution, Faraday Discuss., 103, 227–244.CrossRefPubMedGoogle Scholar
  8. 8.
    Basak, P., and Bhattacharyya, M. (2013) Intrinsic tryptophan fluorescence and related energy transfer in leghemoglobin isolated from Arachis hypogea, Turk. J. Biochem., 38, 9–13.Google Scholar
  9. 9.
    Yagi, M., Sakurai, K., Kalidas, C., Batt, C. A., and Goto, Y. (2003) Reversible unfolding of bovine β-lactoglobulin mutants without a free thiol group, J. Biol. Chem., 278, 47009–47015.CrossRefPubMedGoogle Scholar
  10. 10.
    Albani, J. R. (2011) Substructures formed in the excited state are responsible for tryptophan residues fluorescence in β-lactoglobulin, J. Fluoresc., 21, 1683–1687.CrossRefPubMedGoogle Scholar
  11. 11.
    Losytskyy, M. Y., Kovalska, V. B., Varzatskii, O. A., Sergeev, A. M., Yarmoluk, S. M., and Voloshin, Y. Z. (2013) Interaction of the iron (II) cage complexes with proteins: protein fluorescence quenching study, J. Fluoresc., 23, 889–895.CrossRefPubMedGoogle Scholar
  12. 12.
    Puett, D. (1973) The equilibrium unfolding parameters of horse and sperm whale myoglobin: effects of guanidine hydrochloride, urea and acid, J. Biol. Chem., 248, 4623–4634.Google Scholar
  13. 13.
    Zhang, C., Gao, C., Mu, J., Qiu, Z., and Li, L. (2013) Spectroscopic studies on unfolding processes of apo-neuroglobin induced by guanidine hydrochloride and urea, Biomed Res. Int., Article ID 349542; doi: 10.1155/2013/349542.Google Scholar
  14. 14.
    Zaroog, M. S., and Tayyab, S. (2012) Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase, Process Biochem., 47, 775–784.CrossRefGoogle Scholar
  15. 15.
    Dolgikh, D. A., Gilmanshin, R. I., Brazhnikov, E. V., Bychkova, V. E., Semisotnov, G. V., Venyaminov, S. Yu., and Ptitsyn, O. B. (1981) Alpha-lactalbumin: compact state with fluctuating tertiary structure? FEBS Lett., 136, 311–315.CrossRefPubMedGoogle Scholar
  16. 16.
    Ohgushi, M., and Wada, A. (1983) Molten globule state: a compact form of globular proteins with mobile side chains, FEBS Lett., 164, 21–24.CrossRefPubMedGoogle Scholar
  17. 17.
    Arai, M., and Kuwajima, K. (1996) Rapid formation of a molten globule intermediate in refolding of alpha-lactalbumin, Folding Design, 1, 275–287.CrossRefPubMedGoogle Scholar
  18. 18.
    Eftink, M. R. (1994) The use of fluorescence methods to monitor unfolding transitions in proteins, Biophys. J., 66, 482–501.CrossRefPubMedCentralGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2015

Authors and Affiliations

  • Pijush Basak
    • 1
  • Niloy Kundu
    • 2
  • Rudradip Pattanayak
    • 1
  • Maitree Bhattacharyya
    • 1
    Email author
  1. 1.Department of BiochemistryUniversity of CalcuttaKolkataIndia
  2. 2.Department of ChemistryIndian Institute of TechnologyKharagpurIndia

Personalised recommendations