Spectrophotometric and Fluorometric Study of the Denaturation of Euglena Cytochrome c-552

Irit Aviram, Charlotte Weissmann

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The denaturation of Euglena gracilis cytochrome c-552 by urea and guanidine-HCl is evidenced by a remarkable enhancement of tryptophan fluorescence and by changes in the absorption spectrum. Comparison of the free energy of unfolding at pH 7.0 with corresponding values reported for horse cytochrome c indicates that cytochrome c-552 is considerably less stable. Spectra of unfolded cytochrome c-552 are pH dependent. pKs of 5.9 in 6 M guanidine and 7.1 in 8 M urea were evaluated for a spin transition, which may be assigned to deprotonation of the single histidyl residue of cytochrome c-552. The alkaline-denatured forms are of low spin and lack the 695-nm absorption band; the acid-denatured forms exhibit spectra characteristic of a high-spin iron. Comparison of the denaturation curves at the alkaline and acid ends of the transition indicates that the alkaline unfolding, leading to the formation of the low-spin denatured form, requires significantly lower concentrations of the denaturing agents, especially in the case of urea. Opposite results were obtained for horse cytochrome c. Cytochrome c-552 possesses a remarkable stability towards acid pH. Iodide quenching studies reveal heterogeneous accessibility of the two tryptophanyl residues toward the quencher at low concentrations of the denaturant and equal exposure at advanced stages of denaturation. These results imply that the unfolding of cytochrome c-552 does not represent a true two-state equilibrium.

Original languageEnglish
Pages (from-to)2020-2025
Number of pages6
Issue number10
StatePublished - 1978


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