An electrochemical assay for the characterization of redox proteins from biological electron transfer chains

A sensitive and quick assay for redox proteins based on electrochemical titrations in a thin-layer electrochemical cell is described. Using a combination of modified-electrode and "mediator-enhanced" electrochemistry, equilibration of the cell volume (4 microliters) with the applied potential allows series of spectra as a function of the potential to be recorded rapidly. A complete redox titration between +500 and -600 mV (vs Ag/AgCl/3 M KCl) in 30-mV intervals takes approximately 2 h. The detection limit of the assay, evaluated for cytochrome c at the alpha-band absorption, is quoted to approximately 100 pmol. The use of this redox assay for the detection of redox-active contaminants in biochemical preparations, for the determination of midpoint potentials of redox enzymes, and for the characterization of complex membrane-bound or soluble redox systems is described.