THERMODYNAMIC CHARACTERIZATION OF CYTOCHROME-C AT LOW PH - OBSERVATION OF THE MOLTEN GLOBULE STATE AND OF THE COLD DENATURATION PROCESS

Several reports have pointed out the existence of intermediate states (both kinetic and equilibrium intermediate) between the native and the denatured states. The molten globule state, a compact intermediate state in which the secondary structure is formed but the tertiary structure fluctuates considerably, is currently being studied intensively because of its possible implication in the folding process of several proteins. We have examined the thermal stability of horse cytschrome c at low pH between 2.0 and 3.2 and different potassium chloride concentrations by absorbance of the Soret band, far and near-ultraviolet circular dichroism (u.V. c.d.) and tryptophan fluorescence using a multidimensional spectrophotometer. The concentration of potassium chloride ranged from 0 m to 0.5 m. The experimental thermal denaturation curves show that: (1) the helical content of cytochrome c remains stable at higher temperature when the concentration of salt is increased; whereas (2) the extent of ordering of the tertiary structure is weakly dependent on salt concentration; and (3) for cytochrome c, the stabilization of the molten globule state is induced by the binding of anions. Other salts such as NaCl, LiCl, potassium ferricyanide (K3Fe(CN)6) and Na2SO4 may also be used to stabilize the molten globule state. The thermodynamic analysis of the denaturation curves of c.d. at 222 nm and c.d. at 282 nm shows that, whereas a two-state (native and denatured) transition is observed at low-salt concentration, the far and near-u.v. c.d. melting curves of cytochrome c do not coincide with each other at high-salt concentration, and a minimum of three different thermodynamic states (IIb, intermediate or IIc, and denatured) is necessary to achieve a sufficient analysis. The intermediate state (called IIc) is attributed to the molten globule state because of its high secondary structure content and the absence of tertiary structure. Therefore, at low pH, cytochrome c is present in at least four states (native, IIb, IIc and denatured) depending on the salt concentration and temperature. The thermodynamic parameters, i.e. the Gibbs free energy differences (ΔG), the enthalpy differences (ΔH), the midpoint temperatures (Tm) of the transition (IIb → intermediate (IIc) → denatured) are determined. We also give estimates of the heat capacity differences (ΔCp) from the temperature dependence of the enthalpy differences. The enthalpy change and the heat capacity difference of the IIc → denatured transition are non-zero. The number of charges (protons or chloride anions) released upon transitions are determined by analysing the pH and chloride anion concentration dependence of the Gibbs free energy. At low salt concentration, these values are in good agreement with a microcalorimetric study and can also explain a cold denaturation process at sub-zero temperature, which was first observed for cytochrome c in the present study. Finally, the phase diagram of cytochrome c at low pH proposed previously by Ohgushi and Wada is re-examined in view of the present data. © 1992.