Mediator-Assisted Continuous-Flow Column Electrolytic Spectroelectrochemical Technique for the Measurement of Protein Redox Potentials. Application to Peroxidase

A mediator-assisted continuous-flow column electrolytic spectroelectrochemical method was designed to study redox properties of proteins. This method is based on a flow-through column electrolytic control of a redox buffer containing relatively high concentrations of mediators and an accelerated reaction of a protein with the mediators to reach equilibrium within the electrolysis time. A small amount of a protein sample is introduced into the electrochemically regulated redox buffer in the mode of flow injection analysis (FIA). The equilibrated redox state of the protein is evaluated by a highly sensitive flow-through photodiode array detector. Reproducible and stable background spectra and the employment of the FIA mode allow precise background subtraction in spectral analysis. This method was successfully applied to determining the redox potentials of horseradish peroxidase (HRP) using potassium hexachloroiridate as a mediator. The absorbance versus the electrode potential curves for the redox reactions among the ferric form, compound II, and compound I of HRP were well interpreted by a Nernstian equation based on the two-step one-electron-transfer model. The advantage and the kinetic aspects of this method are discussed in detailed.