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.
Baymann
F
F
Institut für Biophysik und Strahlenbiologie, Universität Freiburg, Federal Republic of Germany.
Moss
D A
DA
Mäntele
W
W
D001419:Bacteria / Q000201:enzymology*; D003217:Conductometry / Q000379:methods*; D003574:Cytochrome c Group / Q000737:chemistry* / Q000302:isolation & purification; D045362:Cytochromes c2; D004566:Electrodes; D004579:Electron Transport; D012241:Rhodopseudomonas / Q000201:enzymology; D012246:Rhodospirillum / Q000201:enzymology; D013053:Spectrophotometry
D003574:Cytochrome c Group; D045362:Cytochromes c2