A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes

This absorbance plate-reader-based assay is suitable for the examination of monoamine oxidase and copper amine oxidase activities versus numerous substrates. The assay is robust, continuous, rapid, highly quantitative, reasonably sensitive, inexpensive and suitable for automation. In the presence of a suitable amine substrate, amine oxidase enzymes generate hydrogen peroxide, which then drives the peroxidase-dependent oxidation of 4-aminoantipyrine. A subsequent interaction with vanillic acid generates stoichiometric amounts of a red quinoneimine dye, the appearance of which is monitored at 498 nm. An alternative procedure in which vanillic acid is replaced by 2,4-dichlorophenol enhances sensitivity but precludes the measurement of monoamine oxidases due to inhibition of these enzymes by dichlorophenol. Some substrates with low redox potentials, such as catecholamines, are not suitable for inclusion in this assay. A researcher familiar with the procedure can manually generate data for 30 full kinetic curves, composed of ten triplicate points, in 8 h.