MEDIATOR-ASSISTED CONTINUOUS-FLOW COLUMN ELECTROLYTIC SPECTROELECTROCHEMICAL TECHNIQUE FOR THE MEASUREMENT OF PROTEIN REDOX POTENTIALS - APPLICATION TO PEROXIDASE

A mediator-assisted continuous-now column electrolytic spectroelectroc hemical method was designed to study redox properties of proteins. Thi s method is based on a now-through column electrolytic control of a re dox buffer containing relatively high concentrations of mediators and an accelerated reaction of a protein with the mediators to reach equil ibrium within the electrolysis time. A small amount of a protein sampl e is introduced into the electrochemically regulated redox buffer in t he mode of flow injection analysis (FIA). The equilibrated redox state of the protein is evaluated by a highly sensitive flow-through photod iode array detector. Reproducible and stable background spectra and th e employment of the FIA mode allow precise background subtraction in s pectral analysis, This method was successfully applied to determining the redox potentials of horseradish peroxidase (HRP) using potassium h exachloroiridate as a mediator. The absorbance versus the electrode po tential curves for the redox reactions among the ferric form, compound II, and compound I of HRP were well interpreted by a Nernstian equati on based on the two-step one-electron-transfer model. The advantage an d the kinetic aspects of this method are discussed in detailed.