Helicobacter pylori arginase mutant colonizes arginase II knockout mice

Figure 3 The rocF mutant of Helicobacter pylori strain SS1 is devoid of arginase activity. Bacteria were harvested in 0.9% NaCl and ice-bath-sonicated (25% intensity, 2 pulses of 30 s each, with 30 s rests on ice between pulses). Following centrifugation (12 000 g, 2 min, 4°C), supernatants were retained on ice. Equal volumes of extract and 10 mmol/L cobaltous chloride (CoCl₂•6 H₂O, final concentration of 5 mmol/L) were preincubated for 30 min at 50-55°C to activate the enzyme. Arginase buffer (15 mmol/L Tris, pH 7.5, or 15 mmol/L MES, pH 6.0, plus 10 mmol/L L-arginine) was added and incubated at 37°C for 1 h. The reaction was stopped by addition of 750 μL acetic acid, and the color developed by addition of 250 μL ninhydrin (4 mg/mL) at 95°C for 1 h and read at 515 nm. The data are presented in units where 1 U is defined as one pmol L-ornithine/min/mg protein + SD. Details of this assay have been reported previously[23].