A SIMPLE AND SENSITIVE FLUORESCENCE ASSAY FOR MONOAMINE OXIDASE AND DIAMINE OXIDASE

¹ Departments of Pharmacology and Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland

A simple, sensitive fluorometric method is described for the assay of monoamine and dia mine oxidase in vitro. This method is based on the formation of an intensely fluorescent product from homovanillic acid and hydrogen peroxide released during oxidative deamina tion of added substrate. Tissue extracts are incubated in phosphate buffer with horseradish peroxidase and homovanillic acid, after which fluorescence intensity is measured in a spectrophotofluorometer without further manipulation of the samples. The method is comparable in sensitivity to radiometric techniques. Numerous substrates can be employed and the reaction can be continuously monitored. Diamine oxidase from rat small intestine oxidized putrescine, cadaverine, histamine and 1,4- methyl histamine, at rates descending in that order, but appeared to have a greater affinity for histamine than for the other sub strates tested. Monoamine oxidase in rat brain and liver had the same relative activities toward a number of substrates. Tryptamine and tyramine were the most active substrates of those examined whereas benzylamine, octopamine, metanephrine and normetanephrine were less active. 1,4- Methyl histamine was an active substrate both for monoamine oxidase in liver and diamine oxidase in small intestine. However, no activity of rat brain monoamine oxidase toward 1,4- methyl histamine was detectable. Catecholamines and 5- hydroxy tryptamine interfered with fluorophore formation from hydrogen peroxide and could not be used as substrates in this method.