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Effect of temperature and guanidine hydrochloride on ferrocytochrome c at neutral pH

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Abstract

Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6±0.3 °C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by ΔG D at 25 °C, is 59±5 kJ mol−1 (DSC) and 65±6 kJ mol−1 (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 °C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.

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Abbreviations

CD:

circular dichroism

cyt c :

cytochrome c

DSC:

differential scanning calorimetry

ferricyt c :

ferricytochrome c

ferrocyt c :

ferrocytochrome c

GdnHCl:

guanidine hydrochloride

NHE:

normal hydrogen electrode

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Acknowledgements

This work was supported by a research grant from the Slovak Grant Agency VEGA no. 3198. We thank Drs. Jaroslava Bágel’ová and Erik Sedlák for helpful comments, and a reviewer for correcting the English.

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Authors and Affiliations

  1. Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 043 53 , Košice, Slovakia

    Rastislav Varhač, Marián Antalík & Mikuláš Bánó

  2. Department of Biochemistry, Faculty of Sciences, P.J. Šafárik University, Moyzesova 11, 041 54 , Košice, Slovakia

    Marián Antalík

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  1. Rastislav Varhač
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  2. Marián Antalík
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Correspondence to Marián Antalík.

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Varhač, R., Antalík, M. & Bánó, M. Effect of temperature and guanidine hydrochloride on ferrocytochrome c at neutral pH. J Biol Inorg Chem 9, 12–22 (2004). https://doi.org/10.1007/s00775-003-0492-1

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  • DOI: https://doi.org/10.1007/s00775-003-0492-1

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