Ampliflu™ Red (10-Acetyl-3,7-dihydroxyphenoxazine), peroxidase from horseradish, hMAO-A, hMAO-B, H2O2, tyramine hydrochloride, selegiline and moclobemide were purchased from Sigma-Aldrich (Steinheim, Germany) and retained under the suggested conditions by supplier. All pipetting processes were performed using a Biotek Precision XS robotic system (BioTek Instruments, Winooski, VT, USA). Measurements were carried out by a BioTek-Synergy H1 microplate reader based on the fluorescence generated (excitation, 535 nm, emission, 587 nm) over a 30 min period, in which the fluorescence increased linearly.
In the enzymatic assay, three different daily prepared solutions were used. (I) Inhibitor solutions: Synthesized compounds and reference agents were prepared in 2% DMSO in 10−3–10−9 M concentrations (10 mL for each concentration). (II) Enzyme solutions: Recombinant hMAO-A (0.5 U/mL) and recombinant hMAO-B (0.64 U/mL) enzymes were dissolved in the phosphate buffer and final volumes were adjusted to 10 mL. (III) Working solution: Horseradish peroxidase (200 U/mL, 100 μL), Ampliflu™ Red (20 mM, 200 μL) and tyramine (100 mM, 200 μL) were dissolved in the phosphate buffer and final volume was adjusted to 10 mL.
The solutions of inhibitor (20 μL/well) and hMAO-A (100 μL/well) or hMAO-B (100 μL/well) were added to the flat black bottom 96-well micro test plate, and incubated at 37 °C for 30 min. After this incubation period, the reaction was started by adding a working solution (100 μL/well). The mixture was incubated at 37 °C for 30 min and the fluorescence (Ex/Em = 535/587 nm) was measured at 5 min intervals. Control experiments were carried out simultaneously by replacing the inhibitor solution with 2% DMSO (20 μL). To check the probable inhibitory effect of inhibitors on horseradish peroxidase, a parallel reading was performed by replacing enzyme solutions with 3% H2O2 solution (20 mM 100 μL/well). In addition, the possible capacity of the inhibitors to modify the fluorescence generated in the reaction mixture due to non-enzymatic inhibition was determined by mixing inhibitor and working solutions.
The specific fluorescence emission (used to obtain the final results) was calculated after subtraction of the background activity, which was determined from vials containing all components except the hMAO isoforms, which were replaced by phosphate buffer (100 μL/well). Blank, control and all concentrations of inhibitors were analyzed in quadruplicate and inhibition percent was calculated by using following equation:
where FCt2: Fluorescence of a control well measured at t2 time, FCt1: Fluorescence of a control well measured at t1 time, FIt2: Fluorescence of an inhibitor well measured at t2 time, FIt1: Fluorescence of an inhibitor well measured at t1 time. The IC50 values were calculated from a dose-response curve obtained by plotting the percentage inhibition versus the log concentration with the use of GraphPad “PRISM” software (version 5.0, GraphPad Software Inc., La Jolla, CA, USA). The results were displayed as mean ± standard deviation (SD).
In the enzymatic assay, three different daily prepared solutions were used. (I) Inhibitor solutions: Synthesized compounds and reference agents were prepared in 2% DMSO in 10−3–10−9 M concentrations (10 mL for each concentration). (II) Enzyme solutions: Recombinant hMAO-A (0.5 U/mL) and recombinant hMAO-B (0.64 U/mL) enzymes were dissolved in the phosphate buffer and final volumes were adjusted to 10 mL. (III) Working solution: Horseradish peroxidase (200 U/mL, 100 μL), Ampliflu™ Red (20 mM, 200 μL) and tyramine (100 mM, 200 μL) were dissolved in the phosphate buffer and final volume was adjusted to 10 mL.
The solutions of inhibitor (20 μL/well) and hMAO-A (100 μL/well) or hMAO-B (100 μL/well) were added to the flat black bottom 96-well micro test plate, and incubated at 37 °C for 30 min. After this incubation period, the reaction was started by adding a working solution (100 μL/well). The mixture was incubated at 37 °C for 30 min and the fluorescence (Ex/Em = 535/587 nm) was measured at 5 min intervals. Control experiments were carried out simultaneously by replacing the inhibitor solution with 2% DMSO (20 μL). To check the probable inhibitory effect of inhibitors on horseradish peroxidase, a parallel reading was performed by replacing enzyme solutions with 3% H2O2 solution (20 mM 100 μL/well). In addition, the possible capacity of the inhibitors to modify the fluorescence generated in the reaction mixture due to non-enzymatic inhibition was determined by mixing inhibitor and working solutions.
The specific fluorescence emission (used to obtain the final results) was calculated after subtraction of the background activity, which was determined from vials containing all components except the hMAO isoforms, which were replaced by phosphate buffer (100 μL/well). Blank, control and all concentrations of inhibitors were analyzed in quadruplicate and inhibition percent was calculated by using following equation:
where FCt2: Fluorescence of a control well measured at t2 time, FCt1: Fluorescence of a control well measured at t1 time, FIt2: Fluorescence of an inhibitor well measured at t2 time, FIt1: Fluorescence of an inhibitor well measured at t1 time. The IC50 values were calculated from a dose-response curve obtained by plotting the percentage inhibition versus the log concentration with the use of GraphPad “PRISM” software (version 5.0, GraphPad Software Inc., La Jolla, CA, USA). The results were displayed as mean ± standard deviation (SD).
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