Abstract
A simple method has been devised to facilitate ultraviolet (UV) spectrophotometric assay of monoamine oxidase (MAO) activity towards serotonin (5-HT) as a substrate. Validation of the methodology is described in this report. Mitochondria were obtained from the cortex of the hog kidney, of which 1 part was suspended in 3 parts of 0.25 M sucrose-phosphate buffer and used as the enzyme source (Mt, 26 mg protein/ml) . The reaction mixture was comprised of phosphate buffer (pH 7.0, 10 mM), 5-HT hydrochloride (10 mM), and semicarbazone acetate (5 mM) in a final volume of 5 ml; fixed amounts of Mt for the time-course study, or varied amounts of Mt for the enzyme amountreaction rate study were included as needed. Varied concentrations of harmaline (HM) or pargyline (PG) were added to another mixture with a fixed amount of Mt for inhibition studies. The mixtures were reacted at 37°C and the resulting semicarbazone of 5-hydroxyindole acetaldehyde was extratced into ethyl acetate for UV scanning. The UV extinction (λmax 274 nm) increased linearly with the time-course or the Mt amounts, suggesting the usefulness of the UV measure-ment for monitoring MAO activity. Inhibition of MAO activity was stronger with HM (PI50= 6.8) and weaker with PG (pI50=3.8), suggesting that 5-HT was the substrate for both the A-type and the B-type of MAO. In a comparison study with benzylamine (BZ) as a substrate, the UV spectra (Amax 280 nm) similar to that from the authentic benzaldehyde were recorded and inhibition was produced only by PG (pI50=5.8) . These results agree with the substrate-selectivity and inhibitor-susceptibility findings reported by us on the MAO activity in slices of hog kidney cortex measured by Warburg's manometry, and reported by others with different materials and methods. The usefulness of this method for assaying MAO activities towards 5-HT has thus been verified.
