Spectroscopic studies of enzyme oxidation products of a variety of aromatic amines

Oxidation of o-phenylenediamine by horseradish peroxide in the presence of hydrogen peroxide has been shown to lead to 2,3-diaminophenazine as the principle product, rather than 2,2$\sp\prime$-diaminoazobenzene which was previously suggested. The identification of the product has been confirmed by comparison to the known compound using visible and fluorescent spectroscopy as well as thin layer chromatography. The 2,3-diaminophenazine absorbs in the visible region and can be detected by conventional spectrophotometric techniques. The fluorescence quantum yield was determined to be 0.025 for the 2,3-diaminophenazine at pH 7.0. Other aromatic amines were also tested in the horseradish peroxidase-hydrogen peroxide system to determine if these compounds: would react and would yield fluorescent oxidation products.