Figure 7
Characteristics of purified B. anthracis His 6 -BaRocF. A. Purification of His 6 -BaRocF. His6-BaRocF was purified as described in Methods and the flow-through (FT), washes (W) and elutions (E) analyzed by Coomassie blue-stained SDS-PAGE. Intervening lanes between W7 and E1 have been cropped out. B and C. Purified His 6 -BaRocF displays optimal catalytic activity with nickel. The purified enzyme was assayed with 5 mM of the appropriate metal after heat activation at pH 6.3 (B) or pH 9.0 (C) in Bis Tris Propane [BTP]-buffered arginine. At least two experiments were conducted for each sample in triplicate. Data are presented as mean arginase activity (nmol L-orn/min/mg protein) ± standard deviation. No Metal, assayed without metal. D. Effect of heat activation on the activity of His 6 -BaRocF. The purified arginase samples were assayed with or without heat activation (50–55°C or on ice for 30 minutes, respectively) in the absence (No Metal) or presence of 5 mM metal (CoCl2, MnSO4, or NiCl2) at pH 6.0 (in MES-buffered arginine) or 9.0 (Tris-buffered arginine). There was no evidence of a buffer effect on activity (data not shown). At least two experiments were conducted in triplicate for each sample. Data are presented as mean arginase activity (nmol L-orn/min/mg protein) ± standard deviation.