ABSTRACT
The picture became clearer when experiments were conducted in which cyclic voltammograms were obtained immediately on introducing a solution of cytochrome c into a cell with an indium oxide working electrode. There was a marked initial time dependence with a final stable response exhibiting electron transfer kinetics that was much more irreversible [43]. Following the suggestion of a collaborator, Jan F.Chlebowski, the now-obvious move to chromatographically purify the cytochrome c sample was made with profound results. Figure 3 shows a set of cyclic voltammograms and derivative cyclic voltabsorptammograms for cytochrome c at indium oxide. The Tris/cacodylic acid buffer was used in this work because there is minimal anion binding to the positively charged lysine residues on the reaction surface of cytochrome c [43]. These responses show no time-dependent loss of response and are stable for hours at indium oxide when the sample is purified chromatographically. The optical results are shown compared with calculated responses, and the agreement is excellent. As will be described later, lyophilized samples contain oligomeric forms of cytochrome c that foul solid electrode surfaces, and the samples used in this work also contained deamidated forms of the protein. A very small amount of impurity is adequate to completely foul a solid electrode, a point commonly known in the electrochemical community, but this is the first report in which importance of this simple principle is described for studies of proteins. Nevertheless, much work continues on protein samples of unknown impurity, accompanied in some cases by convoluted mechanistic explanations that aim to explain results, when the results are simply corrupted by fouled electrodes and have nothing to do with the protein under study.
