Stability of yeast iso‐1‐ferricytochrome c as a function of pH and temperature

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Abstract
Absorbance-detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2-state model. Values for free energy (ΔGD), enthalpy (ΔHD), and entropy (ΔSD) of denaturation were determined as functions of pH and temperature. The value of ΔGD at 300 K, pH 4.6, is 5.1 ± 0.3 kcal mol−1. The change in molar heat capacity upon denaturation (ΔCp), determined by the temperature dependence of ΔHD as a function of pH (1.37 ± 0.06 kcal mol−1 K−1), agrees with the value determined by differential scanning calorimetry. pH-dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a ΔHD of 1.30 kcal mol−1 residue−1 at 373.6 K and a ΔSD of 4.24 cal mol−1 K−1 residue−1 at 385.2 K. These values agree with those for other single-domain, globular proteins.