Protein-metal ion interactions play an important role in many areas of biological chemistry, partly because they can bind to and stabilise protein surface domain structure in the specific conformations that are involved in key molecular recognition events. Metal ions may interact with proteins and peptides to sustain or modify the secondary, tertiary and quaternary structures and/or protein and peptide functions. In addition to the metal binding stoichiometry it is important to know whether the macromolecule exists in its apo or holo form, whether bound ligands of endogenous origin, if present, can be or have been removed, or if the peptide or protein is susceptible to metal ion specificity.

The interaction of cytochrome c with a range of biologically essential (Ca²⁺, Mg²⁺, Zn²⁺, Ni²⁺ and Co²⁺) and toxic (MeHg⁺, Cd²⁺ and Pb²⁺) metal ions has been investigated by electrospray mass spectrometry and cyclic voltammetry at a 4,4' bipyridyl disulfide modified gold electrode. The comparison of data obtained by electrospray mass spectrometry and voltammetry was achieved by compromising solvent systems and electrolyte concentrations under which these techniques are commonly performed. The most compatible solvent/electrolyte system was found to be 65:35 water:isopropanol, with 5 mM ammonium acetate for electrospray mass spectrometry or 80 mM ammonium acetate for voltammetric analysis.

Increasing the concentration of acetic acid in the electrospray solution caused the spectral profile to move to a lower m/z, indicating an increased charge density for cytochrome c. With the electrochemical solution, a slight positive shift in the reversible formal potential was observed. This too is indicative of an increase in the charge density of cytochrome c in the vicinity of the heme group, resulting in destabilisation of the Fe(III) oxidation state.

As the metal salt concentrations were increased the reversible formal potential did not alter, although the shape of the voltammogram changed from peak shaped to sigmoidal. This shape change is attributed to excess positively charged metal ions blocking some of the electro-active sites on the electrode surface, resulting in radial diffusion to the remaining active sites. The addition of metal salts to the electrospray solution produced metal ion adducts, the number of metal ions binding to the protein increasing with metal salt concentration. However, this adduction did not alter the overall charge of the protein, which explains why the voltammogram reversible potential was unaffected by the addition of metal salts.

Electrospray mass spectrometry and cyclic voltammetry showed that the heme group was neither lost nor replaced by any of the added metal ions. No preferential binding to either the Fe(III) or Fe(II) oxidation states was observed.