Skip to main content
SpringerLink
Log in

pH-induced conformational isomerization of leghemoglobin from Arachis hypogea

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

Abstract

The pH dependence of proteins is related to the thermodynamic stability and electrostatic interactions in the native state of a protein. Here we report the pH-induced conformational transition of the heme protein leghemoglobin (Lb) isolated from root nodules of the leguminous plant Arachis hypogea. Unlike the other heme proteins myoglobin, hemoglobin, and cytochrome c, the structural characteristics and interactions of Lb is almost unknown, though its functional importance is already established since it binds oxygen to maintain the environment for N2 fixation. We investigated pH-induced unfolding of this protein and identified a number of conformational isomers using multiple fluorescence observables as a function of pH titration. We have characterized the acid- and base-induced conformational transitions among the structural states over the pH range 2–11. Depending on the solution conditions, Lb can exist in one of three phases: pH 2, 3, 4; pH 5, 6, 7; pH 8, 9, 10. The secondary structure as revealed by CD spectroscopy indicated the maximum percentage of α-helix to be present at pH 7, where the structure of Lb is also most rigid according to fluorescence anisotropy experiments. The fluorescence lifetime of tryptophan was observed to be maximum at pH 10 and minimum at pH 6, suggesting unfolding transitions of Lb. Thus, alteration of the microenvironment of the globin moiety during pH transition ultimately leads to the conformational change of this monomeric protein Lb.

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

a.a.:

amino acid residues

CD:

circular dichroism

Lb:

leghemoglobin

References

  1. Gros, G., Wittenberg, A. B., and Jue, T. (2010) Myoglobin old and new clothes: from molecular structure to function in living cells, J. Exp. Biol., 213, 2713–2725.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Atanasov, B. P., and Zhizheskaia, G. Ia. (1975) Acid-alkaline equilibrium of the ferri-leghemoglobin of the lupine (Lupinus luteus L.). Spectral studies, Mol. Biol. (Moscow), 9, 491–501.

    CAS  Google Scholar 

  3. Krasnobaeva, N. N., and Atanasov, B. P. (1978) Lupine leghemoglobin affinity to ligands. The effect of pH and buffer nature, Mol. Biol. (Moscow), 12, 1239–1245.

    CAS  Google Scholar 

  4. Fuchsman, W. H., and Appleby, C. A. (1979) CO and O2 complexes of soybean leghemoglobin: pH effects upon infrared and visible spectra. Comparisons with CO and of myoglobin and hemoglobin, Biochemistry, 18, 1309–1321.

    Article  CAS  PubMed  Google Scholar 

  5. Voet, D., and Voet, G. J. (2008) Fundamentals of Biochemistry, 3rd Edn., John Wiley & Sons.

    Google Scholar 

  6. Ellis, P. J., Appleby, C. A., Guss, J. M., Hunter, W. N., Ollis, D. L., and Freeman, H. C. (1997) Structure of ferric soybean leghemoglobin a nicotinate at 2.3 Å resolution, Acta Cryst., D53, 302–310.

    CAS  Google Scholar 

  7. Harutyunyan, E. H., Safonova, T. N., Kuranova, I. P., Popov, A. N., Teplyakov, A. V., Obmolova, G. V., Rusakov, A. A., Vainshtein, B. K., Dodson, G. G., and Wilson, J. C. (1995) The structure of deoxy- and oxy-leghemoglobin from lupine, J. Mol. Biol., 251, 104–115.

    Article  CAS  PubMed  Google Scholar 

  8. Tzoneva, R., and Michonova-Alexova, E. I. (1998) A calorimetric study of pH-dependent thermal unfolding of leghemoglobin from soybean, BBA, Sec. Bioenerg., 1364, 420–424.

    CAS  Google Scholar 

  9. Boulton, M., Rozanowska, M., and Rozanowska, B. (2001) Retinal photodamage, J. Photochem. Photobiol. B. Biol., 64, 144–161.

    Article  CAS  Google Scholar 

  10. Appleby, C. A., Nicola, N. A., Hurrel, J. G. R., and Leach, S. J. (1975) Characterization and improved separation of soybean leghemoglobins, Biochemistry, 14, 4444–4450.

    Article  CAS  PubMed  Google Scholar 

  11. Navascues, J., Perez-Rontome, C., Gay, M., Marcos, M., Yang, F., Walker, F. A., Desbois, A., Abian, J., and Becana, M. (2012) Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules, PNAS, 109, 2660–2665.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. 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 

  13. Ingersoll, M. C., and Strollo, C. M. (2007) Steady state fluorescence anisotropy to investigate flavonoids binding to proteins, J. Chem. Educ., 84, 1313–1315.

    Article  CAS  Google Scholar 

  14. 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.

    Article  CAS  PubMed  Google Scholar 

  15. Albani, J. R. (2011) Sub-structures formed in the excited state are responsible for tryptophan residues fluorescence in β-lactoglobulin, J. Fluoresc., 21, 1683–1687.

    Article  CAS  PubMed  Google Scholar 

  16. Lakowicz, J. R. (1999) Principle of Fluorescence Spectroscopy, 2nd Edn., Kluwer, New York.

    Book  Google Scholar 

  17. 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.

    Article  CAS  PubMed  Google Scholar 

  18. Zaidi, N., Ahmad, E., Rehan, M., Rabbani, G., Ajmal, M. R., Zaidi, Y., Subbarao, N., and Khan, R. H. (2013) Biophysical insight into furosemide binding to human serum albumin: a study to unveil its impaired albumin binding in uremia, J. Phys. Chem. B, 117, 2595–2604.

    Article  CAS  PubMed  Google Scholar 

  19. Correa, D. H. A., and Ramos, C. H. I. (2009) The use of circular dichroism spectroscopy to study protein folding, form and function, Afr. J. Biochem. Res., 3, 164–173.

    CAS  Google Scholar 

  20. Ahmad, B., Parveen, S., and Khan, R. H. (2006) Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site, Biomacromolecules, 7, 1350–1356.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  22. Rami, B. R., and Udgaonkar, J. B. (2001) pH-jumpinduced folding and unfolding studies of barstar: evidence for multiple folding and unfolding pathways, Biochemistry, 40, 15267–15279.

    Article  CAS  PubMed  Google Scholar 

  23. McPhie, P. (1975) pH dependence of the thermal unfolding of ribonuclease A, Biochemistry, 11, 879–883.

    Article  Google Scholar 

  24. Bhattacharya, M., Jain, N., Bhasne, K., Kumari, V., and Mukhopadhyay, S. (2011) pH-induced conformational isomerization of bovine serum albumin studied by extrinsic and intrinsic protein fluorescence, J. Fluoresc., 21, 1083–1090.

    Article  CAS  PubMed  Google Scholar 

  25. Ortiz de Montellano, P. R. (2009) Hemes in biology, in Wiley Encyclopedia of Chemical Biology, John Wiley & Sons, Inc., pp. 240–249.

    Google Scholar 

  26. Furuyama, K., Kaneko, K., and Vargas, P. D. (2007) Heme as a magnificent molecule with multiple missions: heme determines its own fate and governs cellular homeostasis, Tohoku J. Exp. Med., 213, 1–16.

    Article  CAS  PubMed  Google Scholar 

  27. Padmanaban, G., Venkateswar, V., and Rangarajan, P. N. (1989) Haem as a multifunctional regulator, Trends Biochem. Sci., 14, 492–496.

    Article  CAS  PubMed  Google Scholar 

  28. Smerdon, S. J., Krzywda, S., Wilkinson, A. J., Brantley, R. E., Jr., Carver, T. E., Hargrove, M. S., and Olson, J. S. (1993) Serine 92 (F7) contributes to the control of heme reactivity and stability in myoglobin, Biochemistry, 32, 5132–5138.

    Article  CAS  PubMed  Google Scholar 

  29. Olson, J. S., Mathews, A. J., Rohlfs, R. J., Springer, B. A., Egeberg, K. D., Sligar, S. G., Tame, J., Renaud, J. P., and Nagai, K. (1988) The role of the distal histidine in myoglobin and hemoglobin, Nature, 336, 265–266.

    Article  CAS  PubMed  Google Scholar 

  30. Quillin, M. L., Arduini, R. M., Olson, J. S., and Phillips, G. N., Jr. (1993) High-resolution crystal structures of distal histidine mutants of sperm whale myoglobin, J. Mol. Biol., 234, 140–155.

    Article  CAS  PubMed  Google Scholar 

  31. Grunwald, E. W., and Richards, M. P. (2006) Studies with myoglobin variants indicate that released hemin is the primary promoter of lipid oxidation in washed fish muscle, J. Agric. Food Chem., 54, 4452–4460.

    Article  CAS  PubMed  Google Scholar 

  32. Hargrove, M. S., and Olson, J. S. (1996) The stability of holomyoglobin is determined by heme affinity, Biochemistry, 35, 11310–11318.

    Article  CAS  PubMed  Google Scholar 

  33. Grandori, R., Schwarzinger, S., and Muller, N. (2000) Cloning, overexpression and characterization of micromyoglobin: a minimal heme-binding fragment, Eur. J. Biochem., 267, 1168–1172.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India

    P. Basak, R. Pattanayak, S. Nag & M. Bhattacharyya

Authors
  1. P. Basak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. Pattanayak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Nag
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. Bhattacharyya
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Bhattacharyya.

Additional information

Published in Russian in Biokhimiya, 2014, Vol. 79, No. 11, pp. 1539–1547.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Basak, P., Pattanayak, R., Nag, S. et al. pH-induced conformational isomerization of leghemoglobin from Arachis hypogea . Biochemistry Moscow 79, 1255–1261 (2014). https://doi.org/10.1134/S0006297914110133

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297914110133

Key words

Search

Navigation