Recombinant horseradish peroxidase - and cytochrome c-based two-electrode system for detection of superoxide radicals

The reliable detection of a superoxide anion radical O2− is complicated by its spontaneous dismutation reaction to H2O2 at acidic pHs. To simultaneously detect both O2− and H2O2 produced in the course of its spontaneous dismutation, an electrochemical two-electrode system based on cytochrome c (cyt c) and recombinant horseradish peroxidase (rHRP) was applied. Therewith, a limited applicability of the cyt c system for the reliable monitoring of O2− in acidic and neutral solutions was shown. It was demonstrated that both the reaction of O2− dismutation to H2O2 and the reaction between the formed H2O2 and O2− chemically decrease the amount of the initially present O2−, decreasing the sensitivity and reliability of the electrochemical detection at acidic pH. However, by appropriately varying solution pH, the concentration of O2− initially injected in the system can be estimated from the analysis of calibration curves for H2O2 obtained with highly sensitive rHRP-modified electrode system at pH 6.0 and 7.0.