The peroxidase-catalyzed oxidation of 3,3;,5,5;-tetramethylbenzidine (TMB), ortho-phenylenediamine (PDA), and 5-aminosalicylic acid (5-ASA) is significantly accelerated in the presence of 2-aminothiazole (AT) and melamine (MA), and an increase in their concentrations is associated with a parallel increase in the k(cat) and K(m) values for TMB and PDA. The activation of the peroxidase-catalyzed oxidation of TMB and PDA is quantitatively characterized by a coefficient (degree) alpha (M(-1)) which significantly depends on pH in the range 6.2-6.4, 6.4-7.4, and 6.0-7.4 for the TMB-AT, TMB-MA, and PDA-MA pairs, respectively. An increase in the coefficient alpha with increase in pH confirms nucleophilicity of activation of the peroxidase-catalyzed oxidation of the aromatic amines in the presence of AT and MA. Under optimal conditions the coefficients alpha for the TMB-AT, PDA-AT, TMB-MA, and PDA-MA pairs vary in the limits of (1.90-3.53)*10(3) M(-1).

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Peroxidase-catalyzed oxidation of o-phenylenediamine (PDA) is greatly activated with melamine (MA) in 15 mM phosphate-citrate buffer at pH 6.0-7.4 in a noncompetitive manner: kcat and Km increase in direct proportion to the MA concentration. An extent of the activation is quantitatively characterized with a coefficient alpha (in M-1), which essentially increases along with the rise in pH from 6.0 to 7.4. MA acts as a nucleophilic catalyst in the oxidation process: it most likely affects the peroxidase active site from the distal position of heme. MA non-competitively inhibits the peroxidase oxidation of PDA at pH 4.3, since it completely loses its nucleophilic properties in acidic medium. A rapid, highly accurate, and simple analytical test system based on the kinetics of melamine-activated oxidation of PDA is proposed for the quantitative determination of melamine within the concentration range of 10(-4)-10(-3) M. This test system uses the spectrophotometric determination of the PDA oxidation product at 455 nm.