*4.6. Analysis of Binding of Inhibitor with The Enzymes*

Enzyme-inhibitors mostly produce inhibition of the target enzyme through the formation of an enzyme–inhibitor complex. The formation of the enzyme–inhibitor complex may be accelerated in the presence of a high concentration of the test inhibitor. The property of binding of test compounds to MAO-A or -B was determined by the formation of the enzyme–inhibitor complex by incubation of the enzyme with a high concentration of the test compound. This was followed by extensive equilibrium dialysis of the enzyme–inhibitor complex. Recovery of catalytic activity of MAO-A and -B was determined before and after the dialysis. The MAO-A enzyme (0.2 mg/mL protein) was incubated with each test compound: **1** (10.0 μM), **2** (25.0 μM), **3** (25.0 μM), **5** (100.0 μM) and clorgyline (0.100 μM), in 1 mL of potassium phosphate buffer (100 mM, pH 7.4). After 20 min incubation at 37 ◦C, the reaction was stopped by chilling the tubes in an ice bath. Similarly, the MAO-B enzyme (0.2 mg/mL protein) was incubated with each test compound: **3** (50.0 μM), **4** (50.0 μM), **6** (50.0 μM), and deprenyl (0.500 μM), in 1.0 mL potassium phosphate buffer (100 mM, pH 7.4). After 20 min incubation at 37 ◦C, the reaction was stopped by chilling the tubes in an ice bath. All the samples with enzyme–inhibitor complex were individually dialyzed against potassium phosphate buffer (25 mM; pH 7.4) at 4 ◦C for 16–18 h (including three buffer changes). The control enzyme (without inhibitor) was also run through the same procedure and the activity of the enzyme was determined before and after the dialysis [36].

#### *4.7. Time-Dependent Inhibition of the Enzyme*

To investigate if the binding of the inhibitor with MAO-A and -B followed time-dependent inhibition kinetics, the enzyme was pre-incubated with the inhibitor for different time periods (0–15 min). The compound concentrations used to test time-dependent inhibition were: **1** (0.20 μM), **2** (1.6 μM), **3** (3.0 μM) and **4** (16.0 μM) and clorgyline (0.010 μM), with MAO-A (5.0 μg/mL). The inhibitor concentrations used to test time-dependent inhibition were: **3** (3.0 μM), **4** (1.0 μM), **6** (2.0 μM), and deprenyl (0.070 μM), with MAO-B (12.5 μg/mL). The controls without inhibitors were also run simultaneously. The activities of the MAO-A and -B enzymes were determined as described above and the percentage of enzyme activity remaining was plotted against the pre-incubation time to determine time-dependent inhibition.
