Inhibition of Urease In Vitro

Inhibition of H. pylori ATCC 43504 urease was determined by measuring ammonia production in 96-well plates by the salicylate-hypochlorite method with minor modification.⁵⁹ In brief, the assay mixtures containing 0.25 μg of urease crude (0.04 urease units) in 0.1 mL of the EDTA-sodium phosphate buffer (pH 7.3) and the constituents at different concentrations were preincubated at 37 °C for 90 min with rotation at 50 rpm. An amount of 0.05 mL of urea solution (10 mM) in the sodium phosphate buffer was added to each well, and the plates were incubated in 30 min. Blank and background wells were similarly prepared but with inactive urease by heating at 100 °C in 30 min. Then stop solutions including 0.035 mL of solution A (146% Na salicylate + 0.1% sodium nitroprusside) and 0.065 mL of solution B (1.78% NaOH +11.57% Na citrate + 0.54% NaOCl) were added in sequence. The ammonia released by the urease activity was quantified by measuring the absorbance on the microplate reader at 625 nm. The protein content was determined using a Bradford protein assay kit. BSA was used as a protein standard. Thiourea served as a standard reference inhibitor. One unit of urease is defined as the amount of enzyme that releases 1 μM NH₃ per minute, at 37 °C, pH = 7.3. Each treatment was performed in triplicate and repeated three times in independent experiments.