Optimization of paraoxonase organophosphatase activity assessment in mouse plasma using paraoxon-ethyl

Aim. To select optimal conditions for determining the organophosphatase activity of paraoxonase in mouse blood plasma using paraoxon-ethyl as a substrate. Materials and Methods. The method was modified from a protocol developed for rats. Enzyme activity was assessed by measuring the formation of 4-nitrophenol at 405 nm. The optimal pH was determined within the range of 6.0–10.5 using 50 mM buffer solutions. The reaction mixture contained fixed concentrations of CaCl2, NaCl, and paraoxon-ethyl (1 mM). The reaction was initiated by adding 20 μL of blood plasma. For kinetic analysis, Tris-HCl buffer (pH 8.0) and varying concentrations of paraoxon-ethyl (0–2 mM) were used. Absorbance was measured over 90 seconds using a ULAB-102 spectrophotometer at 405 nm. Enzyme activity was calculated using a molar extinction coefficient of 18290 M⁻¹cm⁻¹, and protein concentration was determined by the Bradford method. Kinetic parameters (Km, Vmax) were calculated using the “KINETICS” software. Results. The highest paraoxonase activity was observed at pH 8.0–9.0. Thus, pH 8.0 was chosen for further assays. The determined Km was 229 ± 22 μM, and Vmax was 0.070 ± 0.009. A paraoxonethyl concentration of 1.5 mM (5–10×Km) ensured substrate saturation. Time-dependence analysis confirmed a linear increase in product formation within the range of 1–50 μL of plasma. A working volume of 15–20 μL was considered optimal. Conclusions. The assay conditions for paraoxonase activity were optimized: pH 8.0, Km = 229 μM, Vmax = 0.070, and a working plasma volume of 15–20 μL.