Bis(4-nitrophenyl) phosphate phosphodiesterase activity assay

Phosphorylated PorX (P-PorX) was prepared by incubating PorX with buffer A supplemented with 20 mM AcP and 10 mM MgCl₂ for 1 h at 37°C. The AcP and MgCl₂ were then removed using Zeba spin desalting columns equilibrated with buffer A. The phosphodiesterase reaction was initiated by adding 50 μl 2 μM P-PorX (diluted in a test buffer, see below) to 50 μl 10 mM bis(4-nitrophenyl)phosphate (bis- pNPP, Merck) and monitoring the release of the reaction product (p-nitrophenol) for 90 min at 37°C by measuring the absorbance at 405 nm on a SpectraMAX microplate reader (Molecular Devices). The assay was performed in triplicate. For activity pH screening, P-PorX was incubated in 150 mM NaCl, 3 μM ZnCl₂, 100 mM Tris, 50 mM MES, 50 mM acetic acid (pH 5.0–9.5) or CAPS buffer (pH 9.5–10.5) for 30 min at 37°C. For metal screening, P-PorX was incubated in buffer A containing 0.5 mM ZnCl₂, CuCl₂, MnCl₂, MgCl₂ or CaCl₂. PorX was used as a control of the reaction.