An electrospray mass spectrometric and voltammetric study of horse heart cytochrome c in the presence of metal ions

Studies on cytochrome c in the presence of a range of biologically essential (Ca2+, Mg2+, Zn2+, Ni2+ and Co2+) and toxic (MeHg +, Cd2+ and Pb2+) metal ions have been undertaken by both cyclic voltammetry at a 4,4′-bipyridyl disulfide modified gold electrode and by electrospray mass spectrometry. Some bases for comparison of data obtained by the two techniques were achieved by compromising solvent systems and electrolyte (buffer) concentrations with which these techniques are commonly used. The most compatible solvent/electrolyte system for these techniques was found to be 65:35 water:isopropanol with acetic acid, and/or ammonium acetate being added to provide acid, electrolyte and buffer. The electrospray mass spectra of cytochrome c obtained at increased acid concentrations showed a shift in the spectral profile to lower mass-to-charge ratios, indicating an increase in the charge density of cytochrome c. The addition of metal salts to the electrospray solution produced metal ion adduction, the number of metal ions binding to the protein increasing with metal salt concentration. However, the adduction of metal ions to cytochrome c did not alter the overall charge of the protein. Electrospray mass spectrometry and cyclic voltammetry showed that the heme group was neither lost nor replaced by any of the added metal ions. Voltammetric measurements indicate that no preferential binding of metal ions to either the Fe(III) or Fe(II) oxidation states occurs, since the reversible potential obtained from voltammetric measurements did not change following the metal salt additions. © 1998 Elsevier Science S.A.