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.
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Abbreviations
- Cyt c :
-
cytochrome c
- GdnHCl:
-
guanidium hydrochloride
- Hb:
-
hemoglobin
- Lb:
-
leghemoglobin
- Mb:
-
myoglobin
References
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.
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.
Appleby, C. A. (1969) Properties of leghemoglobin in vivo and its isolation as ferrous oxyleghemoglobin, Biochim. Biophys. Acta, 188, 222–226.
Aviram, I., and Weissmann, C. (1978) Spectrophotometric and fluorometric study of the denaturation of Euglena cytochrome c552, Biochemistry, 10, 2020–2025.
Schechter, A. N., and Epstein, C. J. (1968) Spectral studies on the denaturation of myoglobin, J. Mol. Biol., 35, 567–589.
Otting, G., Liepinsh, E., and Wuthrich, K. (1991) Protein hydration in aqueous solution, Science, 254, 974–980.
Denisov, V. P., and Halle, B. (1996) Protein hydration dynamics in aqueous solution, Faraday Discuss., 103, 227–244.
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.
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.
Albani, J. R. (2011) Substructures formed in the excited state are responsible for tryptophan residues fluorescence in β-lactoglobulin, J. Fluoresc., 21, 1683–1687.
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.
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.
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.
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.
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.
Ohgushi, M., and Wada, A. (1983) Molten globule state: a compact form of globular proteins with mobile side chains, FEBS Lett., 164, 21–24.
Arai, M., and Kuwajima, K. (1996) Rapid formation of a molten globule intermediate in refolding of alpha-lactalbumin, Folding Design, 1, 275–287.
Eftink, M. R. (1994) The use of fluorescence methods to monitor unfolding transitions in proteins, Biophys. J., 66, 482–501.
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Published in Russian in Biokhimiya, 2015, Vol. 80, No. 4, pp. 544–555.
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Basak, P., Kundu, N., Pattanayak, R. et al. Denaturation properties and folding transition states of leghemoglobin and other heme proteins. Biochemistry Moscow 80, 463–472 (2015). https://doi.org/10.1134/S0006297915040100
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DOI: https://doi.org/10.1134/S0006297915040100