DETERMINATION OF MONOAMINE OXIDASE IN RAT BRAIN BY ELECTRON SPIN RESONANCE TECHNIQUES

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Abstract
—The recent development of spin‐labeling techniques has permitted its application to the measurement of the concentration of rat brain mitochondrial monoamine oxidase (MAO) and detection of possible multiple forms of this enzyme. Spin‐labeled p‐hydroxyamphetamine (SHA) was used as a probe of the active site of the enzyme. The binding of SHA to MAO was monitored by measuring the intensity of the × band ESR spectra obtained with a Varian V‐4502 ESR spectrometer. The measured volume of each sample was about 0·02 ml and time required for each measurement was 5 min. In the presence of MAO, a linear relationship was observed between spectral intensity and the concentration of the spin‐label in a range of 4 × 10−6m to 10−3m. A plot of the amount of enzyme‐inhibitor complex formed against the concentration of SHA suggested the existence of at least three different affinities of SHA to the possible multiple forms of MAO. Measurements made using a constant concentration of the spin‐label (3·21 × 10−5m) resulted in a calibration curve with two plateaus when the concentration of the enzyme‐inhibitor complex was plotted against the apparent concentration of MAO. These data also suggested that spin‐labeled hydroxyamphetamine had distinctly different affinities toward the multiple forms of MAO that were present in the partially purified preparation employed. Measurements made with constant concentrations of SHA and MAO but with varying temperatures (4‐56°C) resulted in a curve with two plateaus which also suggested the existence of at least three different binding affinities of the enzyme preparation for the inhibitor. This ESR technique is simple, rapid, accurate and fairly sensitive. It was also applied to the measurement of MAO in human platelets.