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.
3,3′,5,5′-Tetramethylbenzidine dihydrochloride hydrate has also been used as a horseradish peroxidase substrate in enzyme linked immunosorbent assay and reverse dot blot (RDB).
Convenient water-soluble form of TMB for determination of peroxidase.
3,3',5,5'-Tetramethylbenzidine dihydrochloride hydrate is a chromophore substrate in ELISA assays. It is a noncarcinogenic substitute for benzidine.
3,3′,5,5′-Tetramethylbenzidine dihydrochloride hydrate (TMB-d) is a sensitive chromogen substrate for peroxidase. It is non- carcinogenic, hence, it can be used as a safe substitute for benzidine. TMB-d is useful in quantifying hemoglobin and in staining myeloperoxidase-containing granules in granulocytes.
