Effects of Specific Anion-Protein Binding on the Alkaline Transition of Cytochrome c

The thermodynamic parameters of the alkaline transition of beef heart ferricytochrome c have been measured through direct electrochemistry experiments carried out at variable pH and temperature in the presence of different sulfate concentrations. Sulfate is known to bind specifically to cytochrome c in a sequential manner at two surface sites. The effects of such a specific binding reflect on the thermodynamics of the transition and can be satisfactorily interpreted within the frame of the Debye-Hückel theory with simple electrostatic considerations. In particular, the increase in the thermodynamic p K a values (extrapolated to I = 0) upon sulfate binding turns out to be a fully enthalpic effect which can be accounted for by considering the coulombic effects of the formation of ionic couple(s) on the protein surface. This study also shows that the apparent p K a values at finite ionic strength are only moderately affected by the nature of the anion in solution, and differences tend to vanish at high ionic strength.