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).
1 The completely oxidized form (diimine) absorbs at 450 nm (ε = 59,000) and is formed by two sequential one-
electron oxidations of TMB.
1,2 Thus the stoichiometry of oxidation is 0.5 mole charge transfer complex (λ
max = 652 nm) or 1 mole of diimine (λ
max = 450 nm) formed (or TMB oxidized) per mole of hydroperoxide reduced by the peroxidase.
