| Assay Method Information | |
| | Xanthine Oxidase (XO) Assay |
| Description: | Xanthine oxidase (XO) assay of 2-(benzylamino)-4-methyl-1,3-thiazole-5-carboxylic acid derivatives 5a-p was evaluated using Bovine milk XO (grade 1, ammonium sulphate suspension, purchased by Sigma Aldrich). UV visible spectrophotometer (EI 2371) was used for measuring uric acid formation at 293 nm at 25 °C under aerobic condition. The reaction mixture containing 1 mL xanthine (0.15 mM), 2.5 mL potassium phosphate buffer (50 mM, pH 7.4) and 0.5 mL of XO solution (0.405 U/mL) was incubated for 5 min at 25 °C. Inhibition of XO activity by various inhibitors was measured by following the decrease in the uric acid formation at 293 nM at 25 °C. The blank was prepared without enzyme solution. Febuxostat was used as positive control. The enzyme was preincubated for 5 min, with test compounds ranging from 6.25 to 100 µM (six concentrations in triplicate) dissolved in DMSO (1% v/v) (38,39), and the reaction started by the addition of xanthine. DMSO (1% v/v) did not interfere with the enzyme ac |
| Affinity data for this assay | |
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