Sephadex g 25 column pd 10 columns

Helicobacter pylori (ATCC 43504; American Type Culture Collection, Manassas, VA) was grown in brucella broth supplemented with 10% heat-inactivated horse serum for 24 h at 37 °C under microaerobic condition (5% O 2 , 10% CO 2 , and 85% N 2 ). The preparation method of Helicobacter pylori urease by Mao was followed. Briefly, broth cultures (50 mL, 2.0 × 10 8 CFU mL -1 ) were centri-Sheng et al.: Synthesis, Crystal Structures and Urease Inhibition ... fuged (5000 g, 4 °C) to collect the bacteria, and after washing twice with phosphate-buffered saline (pH 7.4), the Helicobacter pylori precipitate was stored at -80 °C. While the Helicobacter pylori was returned to room temperature, and mixed with 3 mL of distilled water and protease inhibitors, sonication was performed for 60 s. Following centrifugation (15,000 g, 4 °C), the supernatant was desalted through SephadexG-25 column (PD-10 columns, Amersham-Pharmacia Biotech, Uppsala, Sweden). The resultant crude urease solution was added to an equal volume of glycerol and stored at 4 °C until used in the experiment. The mixture, containing 25 μL (4U) of Helicobacter pylori urease and 25 μL of the test compound, was preincubated for 3 h at room temperature in a 96-well assay plate. Urease activity was determined for three parallel times by measuring ammonia production using the indophenol method as described by Weatherburn. 9