Activity assays with pNP substrates

pNP-carboxyl esters in various acyl chain lengths (pNP-C2 to C18), pNP-phosphate (pNPP), bis-pNP-phosphate (bis-pNPP), paraoxon-ethyl and parathion-ethyl were purchased from Sigma-Aldrich (Munich, Germany), pNP-phenylphosphonate (pNPPP), and pNP-phosphorylcholine (pNPPC) from Biomol GmbH (Hamburg, Germany). In all, 10 mM stock solutions were prepared in 2-propanol and stored at −20 °C. Standard assays were performed in 200 µL containing 190 µL buffer with 1 mM substrate and 10 µL enzyme solution (0.1 or 1 mg mL⁻¹) and incubated at 90 °C for 30 min (unless otherwise indicated). The effect of different divalent metals (Mg, Ca, Mn, Fe, Co, Ni, Cu, and Zn) on the enzyme activity was studied by adding 1 mM of the corresponding metal chloride salts to the reaction. Reactions were stopped by the addition of 20 µL 2 M Na₂CO₃ and the formation of p-nitrophenolate was measured spectrophotometrically at 405 nm on a Biotek Synergy HT (Bad Friedrichshall, Germany) plate-reader. All assays were performed in triplicate and a buffer control was added. A standard curve with known concentrations of pure p-nitrophenolate was used to determine the extinction coefficient (ε) of the hydrolysis product. Temperature optimum was determined in the range of 40–95 °C. The optimal pH was assayed with different buffers between pH 4 and 10 (0.1 M; pH 4–6: citrate-phosphate buffer; pH 7–8: tris buffer; pH 9–10: carbonate-bicarbonate buffer). For kinetic studies, several substrate concentrations were assayed and aliquots were taken at different time points of the reaction and stored on ice until absorbance was measured. One unit (U) was defined as the amount of protein converting 1 µmol substrate per minute. V_max, K_m, and k_cat were calculated according to Michaelis–Menten kinetics.