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TMB is an aromatic amine that undergoes oxidation by the higher oxidation states of heme peroxidases (compounds I and II) thereby serving as a reducing co-substrate. One electron oxidation of TMB results in a radical cation that forms a charge-transfer complex with the unoxidized compound. This charge transfer complex absorbs at 652 nm (ε = 39,000). The completely oxidized form (diimine) absorbs at 450 nm (ε = 59,000) and is formed by two sequential one-electron oxidations of TMB. Thus the stoichiometry of oxidation is 0.5 mole charge transfer complex (λmax = 652 nm) or 1 mole of diimine (λmax = 450 nm) formed (or TMB oxidized) per mole of hydroperoxide reduced by the peroxidase.

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