The effects of the test compounds on hMAO were investigated using fluorimetric assay [34 (link),35 (link)]. Recombinant hMAO-A and hMAO-B, expressed in BTI-TN-5B1-4 insect cells, horseradish peroxidase type II and p-tyramine hydrochloride were purchased from Sigma Aldrich. Amplex Red was synthesized as previously described [36 ].
Briefly, 50 mM sodium phosphate buffer (pH = 7.4, 0.05 vol.% Triton X-114) containing the compounds or the reference inhibitors and hMAO were incubated at 37 °C for 30 min. The reaction was started by adding Amplex Red (final concentration, 250 µM), horseradish peroxidase (final activity, 1 U/mL), and p-tyramine (final concentration, 1 mM). The increase in fluorescence (λex = 530 nm, λem = 590 nm) was monitored for 30 min at 37 °C. DMSO at a concentration of 1.5% (v/v) was used for the control experiments. For the determination of blank (b), the enzyme was replaced by phosphate-buffered solution. Each measurement was performed in duplicate. Inhibitory potencies were expressed as residual activities (RA), as described under the Cholinesterase assay section.
Briefly, 50 mM sodium phosphate buffer (pH = 7.4, 0.05 vol.% Triton X-114) containing the compounds or the reference inhibitors and hMAO were incubated at 37 °C for 30 min. The reaction was started by adding Amplex Red (final concentration, 250 µM), horseradish peroxidase (final activity, 1 U/mL), and p-tyramine (final concentration, 1 mM). The increase in fluorescence (λex = 530 nm, λem = 590 nm) was monitored for 30 min at 37 °C. DMSO at a concentration of 1.5% (v/v) was used for the control experiments. For the determination of blank (b), the enzyme was replaced by phosphate-buffered solution. Each measurement was performed in duplicate. Inhibitory potencies were expressed as residual activities (RA), as described under the Cholinesterase assay section.