| Assay Method Information | |
| | Urease Inhibition Assay |
| Description: | To understand the binding modes of the compounds given in Table 1, all ligands were docked into the binding site of Urease enzyme. The top ranked conformations of each ligand were saved in a separate database for further evaluation. The docking results well correlated with the experimental results. The reaction mixtures, comprising 25 uL of enzyme (jack bean urease, Sigma aldrich) solution and 55 uL of buffers containing 100 mM urea, were incubated with 5 uL of the test compounds (0.5 mM concentration) at 30 °C for 15 min in 96-well plates. For the kinetics assessment the urea concentrations were changed from 2 to 24 mM. Urease activity was determined by measuring ammonia production using the indophenol method as described by Weatherburn. Briefly, 45 uL of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside (Sigma Aldrich)) and, 70 uL of alkali reagent (0.5% w/v NaOH (Sigma Aldrich) and 0.1% active chloride NaOCl (Sigma Aldrich)) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 uL. The results (change in absorbance per min) were processed by using SoftMax-Pro software (molecular Device, USA). The entire assays were performed at pH 6.8. |
| Affinity data for this assay | |
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