Figure 4.

Denaturation and hydrolysis of diethylphosphorylated AChE in D₂O. A mixture of 2.0 μM paraoxon and 1.4 μM AChE in 100 mM sodium phosphate and 0.1% BSA (buffer) in D₂O (pH 8.0) was incubated for 70 min at 25 °C. A 4-fold excess of buffer in D₂O was added, and 200 μL was applied to a 0.5 mL Sephadex G50 spin column (Fisher) that had been prewashed four times in buffer in D₂O and centrifuged at 1000× g for 1 min. The eluent was cooled to 4 °C, and 60 μL was added either to 640 μL of buffer in D₂O at 25 °C or 640 μL of the same buffer with 2-PAM (to 200 μM) at 25 °C. Aliquots (30 μL) were assayed as in Figure 3 at the times indicated on the x-axis. Assay values v for the solution containing 2-PAM (blue points that correspond to enzyme denaturation) were first fit to Equation (2) with t_aged set to 0 and fixed initial estimates (min⁻¹ for k values) of k_A = 4 × 10⁻⁵, k_H = 4 × 10⁻⁵, k_r = 2 × 10⁻² and E₀ = 0.01 ΔA/min to obtain k_X = 2.2 × 10⁻⁵ and E_tot =0.69 ΔA/min. These values of t_aged, k_A, and E_tot along with k_X = 4 × 10⁻⁵ and k_r = 0 were then fixed into Equation (2) to fit the assay values v for the solution without 2-PAM (green points that reflect enzyme denaturation and diethylphosphorylated enzyme hydrolysis). This fitting gave E₀ = 0.003 ΔA/min and a k_H value of (47 + 3) × 10⁻⁶ min⁻¹ included in Table 1.