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

Nature Protocols volume 1, pages 2498–2505 (2006)Cite this article

Abstract

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.

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Figure 1: Amine-derived hydrogen peroxide and other products in a reaction catalyzed by an amine oxidase enzyme, with subsequent coupling through horseradish peroxidase to generate a colored dye.
Figure 2: Hydrogen peroxide standard curves, with a vanillic-acid-based chromogen, measured at 498 nm (open circles) or a 2,4-dichlorophenol-based chromogen, measured at 510 nm (closed triangles).
Figure 3: Typical data set collected from a single well.
Figure 4: Linear dependence of initial reaction velocities on enzyme concentrations.
Figure 5: Example kinetic plots obtained with recombinant human MAO isozymes.

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Acknowledgements

A.H. thanks the Canadian Institutes of Health Research (Operating Grant MOP77529) and the Faculty of Medicine and Dentistry, University of Alberta, for financial support. We thank Owen Degenhardt for technical assistance. Dedicated to the memory of Dr. Dennis Sharman.

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  1. Department of Pharmacology, 9–70 Medical Sciences Building, University of Alberta, Edmonton, T6G 2H7, Alberta, Canada

    Andrew Holt

  2. Carlsberg Laboratory, Gamle Carlsberg Vej 10, Valby, DK-2500, Denmark

    Monica M Palcic

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Correspondence to Andrew Holt.

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Holt, A., Palcic, M. A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat Protoc 1, 2498–2505 (2006). https://doi.org/10.1038/nprot.2006.402

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