A ONE-STEP FLUOROMETRIC METHOD FOR THE CONTINUOUS MEASUREMENT OF MONOAMINE-OXIDASE ACTIVITY

We have developed a one-step fluorometric method for the measurement o f monoamine oxidase (MAO) activity in 96-well microplates with sensiti vity 10-fold higher than the conventional spectrophotometric assay met hod. This assay is based on the detection of H2O2 in a horseradish per oxidase-coupled reaction using N-acetyl-3,7-dihydroxyphenoxazine (Ampl ex Red), a highly sensitive and stable probe for H2O2. With a single s ampling, this assay is useful for performing both end-point and contin uous measurements of MAO activity. Using a commercially available enzy me, our assay allows the detection of MAO B activity as low as 1.2 x 1 0(-5) U/ml. When applied to crude tissue homogenates, we have been abl e to selectively detect both MAO A and MAO B from cow brain tissue wit h protein content as low as 200 mu g per sample. The potential applica tions of this assay include the measurement of MAO activity in normal and diseased tissues, blood samples, and other biological fluids and t he screening of drugs for the treatment of MAO-mediated diseases. (C) 1997 Academic Press.