A mechanistic study on the thermal unfolding of cytochrome c in presence of 4-chlorobutan-1-ol: Differential scanning calorimetric and spectroscopic approach

Extensive studies have been carried out on thermal unfolding and conformational characterization of cytochrome c in the presence of 4-chlorobutan-1-ol at pH 4.0, 6.0 and 7.0 using micro differential scanning calorimetry, fluorescence, and circular dichroism measurements. Cytochrome c follows a simple two-state reversible thermal unfolding in the presence of low concentration (<25 × 10−3 mol dm−3) of the alcohol at pH 4.0 as reflected by equal calorimetric and van't Hoff enthalpies. However, at concentrations higher than 50 × 10−3 mol dm−3, the calorimetric transitions are irreversible and scan rate dependent, thus restricting the application of equilibrium thermodynamics. It has been observed that between 25 × 10−3 mol dm−3 and 50 × 10−3 mol dm−3 alcohol concentration, cytochrome c exists in two conformational states at pH 4.0 following N ⇌ E → U unfolding mechanism, where N ⇌ E transition occurs at 67.6 °C and E → U at 83.0 °C. The spectroscopic results indicate that the protein at 83.0 °C (where the second transition ends) in the presence of the alcohol is in the molten globule state. Quantitative thermodynamic parameters accompanying the thermal denaturation process have been evaluated. 4-Chlorobutan-1-ol has also been observed to induce cooperative thermal transitions in cytochrome c at pH 6.0 and 7.0 whereas in the absence of the alcohol, no thermal transition is observed. From the scan rate dependent irreversible thermal transitions, an average activation energy of 136 ± 2 kJ mol−1 has been calculated.