Methods Materials Standard H. pylori strains SS1 and ATCC 43504 were obtained from Shanghai Institute of Digestive Disease. E. coli strain BL21 (DE3) was purchased from Stratagene. All chemicals were of reagent grade or ultra-pure quality, and commercially available. HpFabZ enzymatic inhibition assay The expression, purification and enzymatic inhibition assay of HpFabZ enzyme were performed according to the previously published approach [7, 8] with slight modification. The compounds dissolved in 1% DMSO (Dimethyl sulfoxide) were incubated with the enzyme for 2 hours before the assay started. The IC50 value of Emodin was estimated by

fitting the inhibition data to a dose-dependent curve using a logistic derivative equation. The inhibition type of Emodin www.selleckchem.com/products/nct-501.html against HpFabZ was determined in the presence of varied inhibitor concentrations. After 2h-incubation, the reaction was started by the addition of crotonoyl-CoA. The K i value Selleckchem GM6001 was obtained from Lineweaver-Burk double-reciprocal plots and subsequent

secondary plots. Surface Plasmon Resonance (SPR) technology based binding assay The binding of Emodin to HpFabZ was analyzed by SPR technology based Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). All the experiments were carried out using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA and 0.005% surfactant P20) as running buffer with a constant flow rate of 30 μL/min at 25°C. HpFabZ protein, which was diluted in 10 mM sodium acetate buffer (pH 4.13) to a final concentration of 1.3 μM, was covalently immobilized on the hydrophilic carboxymethylated dextran matrix of the CM5 sensor chip (BIAcore) using standard primary before amine coupling procedure. Emodin was dissolved in the running buffer with different concentrations Selleck BAY 11-7082 ranging from 0.625 to 20 μM. All

data were analyzed by BIAevaluation software, and the sensorgrams were processed by automatic correction for nonspecific bulk refractive index effects. The kinetic analyses of the Emodin/HpFabZ binding were performed based on the 1:1 Langmuir binding fit model according to the procedures described in the software manual. Isothermal titration calorimetry (ITC) technology based assay ITC experiments were performed on a VP-ITC Microcalorimeter (Microcal, Northampton, MA, USA) at 25°C. HpFabZ was dialysed extensively against 20 mM Tris (pH 8.0), 500 mM NaCl and 1 mM EDTA at 4°C. Appropriate concentration of Emodin was prepared from a 50 mM stock in DMSO, and corresponding amount of DMSO (25%) was added to the protein solution to match the buffer composition. The reference power was set to 15 μCal/sec and the cell contents were stirred continuously at 300 rpm throughout the titrations.