Determination of the molar absorptivities of phenothiazine cation radicals generated by oxidation with hydrogen peroxide/peroxidase.

Phenothiazines are used as antipsychotic drugs and as reagents to determine microamounts of hemoglobin in biological fluids and tissues. Several agents cause the oxidation of phenothiazines to chromophoric cation radicals, whose stability may be related with their biological action. Enzymes and proteins with peroxidase activity catalyze the oxidation by H2O2 of phenothiazines to their corresponding cation radicals, which suffer a nonenzymatic breakdown. The instability of these cation radicals makes the determination of their respective molar absorptivities very difficult. These properties, however, have been determined for a few phenothiazine cation radicals by cumbersome or unreliable procedures. In this paper a new method is proposed and applied to six different phenothiazines oxidized with H2O2/peroxidase. The method involves the stoichiometric exhaustion of H2O2, under assay conditions which yield a fast enzymatic formation of phenothiazine cation radicals and which slow down their nonenzymatic breakdown. This method may be useful for quantitative studies on the enzymatic activity and the reaction mechanism of the oxidation of a number of phenothiazines catalyzed by different types of peroxidase, as well as by proteins with peroxidase activity, such as hemoglobin.