The reduction mechanism of cytochrome c and methemoglobin on the mercury electrode

Abstract

Using AC polarography on a stationary mercury electrode and transferring the electrode covered with an adsorbed protein into another protein solution possessing a greater electrochemical activity, we established that the exchange of the protein from the first layer does not occur and that the same monolayer of flattened protein develops both at large and small concentrations. Using the same technique of layer—by—layer adsorption of two different proteins, but enlarging the mercury drop size in the buffer, we studied adsorption in the second layer. The mercury drop enlargment permits the second layer to move over onto the electrode surface, so that it becomes visible. It was found that adsorption in the second layer is reversible and that no denaturating processes occur in it. The data obtained about the nature of the protein adsorption were used to analyse the reduction mechanism of cytochrome, methemoglobin and other heme—containing proteins. It was shown that the reduction current of oxidized cytochrome and methemoglobin depends linearly on protein concentration up to 7–10 mg/cm 3. Ten—fold excess of BSA, globulin or ribonuclease at the total concentration of 40 mg/cm 3 does not lead to any change in the cytochrome wave height. The data obtained made it possible to formulate a hypothesis explaining the protein reduction mechanism. The first portions of protein denaturate irreversibly on the electrode surface, forming a monolayer of flattened protein. In the pores of the monolayer reduction of native molecules capable of desorption takes place. This mechanism may be extrapolated to all electrochemical processes occurring both during the reduction of heme—groups and of other electron—active groups in proteins. In a number of cases no polarographic wave was observed due to the strongly irreversible process, as is the csae with the reduction of sulfide groups.