Noncompetitive activation of the peroxidase-catalyzed oxidation of o-phenylenediamine by melamine

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: k(cat) and K-m 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 noncompetitively 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 1 of 10(-4) 10(-3) M. This test system uses the spectrophotometric determination of the PDA oxidation product at 455 nm.