Potentiometric Measurement of Oxidation-Reduction Potentials

The free energy change associated with the oxidation or reduction of a purified flavoprotein is assessed by measuring the oxidation-reduction (redox) potential of the bound flavin. The measurement is made by balancing the partially-reduced flavoprotein against another redox system of known redox potential (,). The reference can be a second redox system in solution, such as a well-characterized dye, in which case the flavoprotein and dye are brought into an equilibrium that can be analyzed spectroscopically. Alternatively, the potential in the solution of partially reduced flavoprotein is balanced against the potential of a standard electro chemical half-cell, such as a mercury/mercuric chloride (calomel) electrode. In the second case, the redox potential of the flavoprotein is probed with an inert metal such as gold or platinum, electrical connection between the flavoprotein and the calomel electrode is completed with a salt bridge made from saturated KCl, and the difference in potential between the partially reduced flavoprotein and the standard electrochemical half-cell is determined with a voltmeter. A value for the redox potential at half-reduction of the flavoprotein is calculated (E M) and expressed in volts with the standard hydrogen electrode as the point of reference (E M=0 V). When E M for one system is less positive (or more negative) than E M for a second system, the first system is the more reducing and the direction of electron transfer will tend to be towards the second system.