The standard potentials of proteins are not commonly measured by the methods described in this chapter because proteins often lose their native structure and function when they react on the surfaces of electrodes. In an alternative method, the oxidized protein is allowed to react with an appropriate electron donor in solution. The standard potential of the protein is then determined from the Nernst equation, the equilibrium concentrations of all species in solution, and the known standard potential of the electron donor. We shall illustrate this method with the protein cytochrome c. The one-electron reaction between cytochrome c, cyt, and 2,6-dichloroindophenol, D, can be followed spectrophotometrically because each of the four species in solution has a distinct colour, or absorption spectrum. We write the reaction as cytox+ Dred ~ cytred+ Dox where the subscripts 'ox' and 'red' refer to oxidized and reduced states,

respectively.

(a) Consider E~yt and E15 to be the standard potentials of cytochrome c and D, respectively. Show that, at equilibrium ('eq'), a plot of ln([Doxlei[DredJeq) versus In ([cytoxJei[cytred]eq) is linear with slope of 1 and y-intercept F(E~yt- Eo)/RT, where equilibrium activities are replaced by the numerical values of equilibrium molar concentrations.

(b) The following data were obtained for the reaction between oxidized cytochrome c and reduced D in a pH 6.5 buffer at 298 K. The ratios [Dox]eq[D [Dox] eq / [Dred]eq 0.00279 0.00843 0.0257 0.0497 0.0748 0.238 0.534

[Cytox]eq/ [Cytred]eq 0.0106 0.0230 0.08940.197 0.335 0.809 1.39