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The interest in monoamine oxidases A and B (MAO A and B) is due to their central role in regulating the balance of neurotransmitters, both in the central nervous system and in peripheral organs. As validated drug targets for depression and Parkinson's disease, the elucidation of their crystal structures was an essential step to guide drug design investigations. The development of the heterologous expression system of MAO B in Pichia pastoris and the identification of the detergent, buffer, and precipitant conditions allowed to determine the first crystal structure of human MAO B in 2002. A detailed protocol to obtain reproducible MAO B crystals is described.

MAO activity measurement can be monitored by direct peroxidase-free assays following different spectroscopy methods. Typically, these are assays that follow the conversion of different MAO substrates into its corresponding products monitored in either absorbance or fluorescence. Herein, we describe the assays for enzyme activity assessment with MAO B and particularly the MAO A substrate kynuramine, as well as the MAO B substrate benzylamine. Moreover, we also describe MAO activity determination using the tertiary amine substrate allyl amine 1-methyl-4-(1-methyl-1 H-pyrrol-2-yl)-1,2,3,6-tetrahydropyridine (MMTP). These are very useful methods for the investigation of MAO inhibitory activity by molecules known to be HRP-interfering. In the present chapter we demonstrate the application of these methods in MAO activity and Michaelis-Menten curve determinations as well as inhibitory activity experiments.

MAO activity measurement is generally performed following different spectroscopy methods, in most cases using peroxidase as a coupled reaction catalyst. In the presence of horseradish peroxidase (HRP), the assay follows the oxidation of the typical MAO substrate (aromatic amines) which generates hydrogen peroxide as a secondary product. There are several chromogens and fluorogens that, in the presence of hydrogen peroxide, are converted by HRP to detectable products. In the present chapter we describe the spectrophotometric 4-aminoantipyrine assay as well as the fluorogenic assay with the Amplex® Red chemical probe. These methods are applied on MAO activity and Michaelis-Menten curve determinations as well as inhibitory activity experiments.