The horseradish peroxidase-catalyzed oxidation of 3,5,3',5'-tetramethylbenzidine. Free radical and charge-transfer...

Benzidine and related compounds are well known substrates for horseradish peroxidase/H2O2 oxidation. Typically, two different colored products are formed. In this paper, we study the oxidation of 3,5,3',5'-tetramethylbenzidine. The first colored product is a blue charge-transfer complex of the parent diamine and the diimine oxidation product. This species exists in rapid equilibrium with the radical cation. The radical was observed by ESR spectroscopy, and hyperfine splitting constants were determined. Addition of equimolar hydrogen peroxide yields the yellow diimine, which is stable at acid pH. At less than equimolar peroxide, all four species (diamine, radical cation, charge-transfer complex, and diimine) exist in equilibrium. A theoretical analysis of this redox system is presented, including a determination of the extinction coefficients and equilibrium constant for the nonradical species.