| Assay Method Information | |
| | AO Enzyme Assay |
| Description: | Guinea pig AO activity was assayed spectrophotometrically using phenanthridine as a substrate at 322 nm. All spectrophotometric determinations were carried out at 25 °C using a PClinked perkin Elemer Lambda 25 UV/Vis spectrophotometer. The varying substrate concentrations (4-60 µM) were separately added into Sorenson's phosphate buffer (final concentration of 50 mM, pH 7.0) containing 0.1 mM of EDTA. Then, the reaction was started by addition of the enzyme and monitored for up to 2 min. The enzyme was also assayed in the presence of different concentrations of the compounds as potential inhibitors (1-200 µM) and constant concentration of substrate (40 µM) to calculation of IC50 values. The results were compared with the inhibitory effects of 1-100 µM menadione, a specific AO inhibitor. Before enzymatic reaction, all of the solutions were equilibrated at 25 °C, except the enzyme fraction which was kept on ice bath prior to addition to the incubation solution. All compounds were dis |
| Affinity data for this assay | |
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