Paraoxon hydrolysis

Figure 1 Proposed mechanism for paraoxon hydrolysis by organophosphorus hydrolase (X and X are unspecified ligands).

Enzymatic activity was determined spectrophotometrically using Agilent 8453 UV-visible spectroscopy system equipped with a thermostatted cell (Agilent Technology, Germany). The accumulation of / -nitrophenolate anion as product of Paraoxon hydrolysis was monitored at 25°C and 405 run. The amount of enzyme hydrolyzing one pmole of substrate for 1 min at 25°C and pH 10.5 (100 mM Na-carbonate buffer) was considered as one unit of enzymatic activity. 

The N-Hise-e-pHluoriUs-OPH purified by metal-chelating chromatography was characterized in terms of its homogenity and relative catalytic activity in reaction of Paraoxon hydrolysis. It was shown by SDS-PAAG electrophoresis that the homogenity of purified proteins were 95% (Figure 7). The specific activity of N-Hise-e-pHluoriUs-OPH in reaction of... 

Wong, K.Y, Gao, J. The reaction mechanism of paraoxon hydrolysis by phosphotriesterase from combined QM/MM simulations. Biochemistry 2007,46(46), 13352-69. 

OPH catalyzes the hydrolysis of organophosphonates with P-0, P-F, P-S, and P-CN phosphoryl bonds such as those of coumaphos, sarin, VX, and paraoxon. Hydrolysis is facilitated by two metal atoms in the active site and results in the release of two protons (79). In the native dimeric form of the enzyme, zinc is coordinated to histidine residues in the active site. Reconstitution of the apo-entyme with cobalt or cadmium results in higher enzymatic activity than that observed in the zinc-metalloenzyme (20). 

Depending on the metal ion substitution in OpdA, the enzyme displays a high mechanistic flexibility for OP degradation [26]. For the Zn(ll) and Cd(ll) derivatives one kinetically relevant protonation equilibrium was observed in ethyl paraoxon hydrolysis at a low pKa range of around 4—5, which is typical for a... 

The rat LD qS are 13, 3.6 (oral) and 21, 6.8 (dermal) mg/kg. Parathion is resistant to aqueous hydrolysis, but is hydroly2ed by alkah to form the noninsecticidal diethjlphosphorothioic acid and -nitrophenol. The time required for 50% hydrolysis is 120 d ia a saturated aqueous solution, or 8 h ia a solution of lime water. At temperatures above 130°C, parathion slowly isomerizes to 0,%diethyl 0-(4-nitrophenyl) phosphorothioate [597-88-6] which is much less stable and less effective as an insecticide. Parathion is readily reduced, eg, by bacillus subtilis ia polluted water and ia the mammalian mmen to nontoxic 0,0-diethyl 0-(4-aminophenyl) phosphorothioate, and is oxidized with difficulty to the highly toxic paraoxon [511-45-5] diethyl 4-nitrophenyl phosphate d 1.268, soluble ia water to 2.4 mg/L), rat oral LD q 1.2 mg/kg. 

Whole cell OPH activity was measured by following the increase in absorbancy of p-nitrophenol from the hydrolysis of substrate (0.1 mM Paraoxon) at 400 nm (sm = 17,000 M cm ). Samples of culture (1 ml) were centrifuged at 10,000 g and 4 C for 5 min. The cells were washed, resuspended with distilled water, and 100 pi was added to an assay mixture containing 400 pi 250 mM CHES [2-(N-cyclohexylamino)ethane-sulfonic acid] buffer, pH 9.0, 100 pi 1 mM Paraoxon, and 400 pi distilled water. One unit of OPH activity was defined as pmoles Paraoxon hydrolyzed per min. Each value and error bar represents the mean of two independent experiments and its standard deviation. 

FIGURE 2.1 Reaction scheme for OPH hydrolysis of methyl parathion and paraoxon (a) followed by the electrochemical oxidation of p-phenol (b). R and R are ethoxy and methoxy and X is O and S in paraoxon and methyl parathion, respectively. 

Although the inhibition-based biosensors are sensitive, they are poor in selectivity and are rather slow and tedious since the analysis involves multiple steps of reaction such as measuring initial enzyme activity, incubation with inhibitor, measurement of residual activity, and regeneration and washing. Biosensors based on direct pesticide hydrolysis are more straightforward. The OPH hydrolyzes ester in a number of organophospho-rus pesticides (OPPs) and insecticides (e.g. paraoxon, parathion, coumaphos, diazinon) and chemical warfare agents (e.g. sarin) [53], For example, OP parathion hydrolyzes by the OPH to form p-nitrophenol, which can be measured by anodic oxidation. Rainina... 

Antibody 15C5 was able to catalyse the hydrolysis of the triester [105] with cat 2.65 x 10 3 min 1 whilst a second antibody from the same immunization programme was later found to hydrolyse the acetylcholinesterase inhibitor Paraoxon [106] with kcat = 1.95 x 10 3min-1 at 25°C (Appendix entry 6.2) (Lavey and Janda, 1996b). Antibody 3H5 showed Michaelis-Menten kinetics and was strongly inhibited by the hapten [104]. It exhibited a linear dependence of the rate of hydrolysis on hydroxide ion concentration, suggesting that 3H5 effects catalysis by transition state stabilization rather than by general acid/base catalysis. 

In both soils and water, chemical and biological mediated reactions transform parathion to paraoxon (Alexander, 1981). Parathion was reported to biologically hydrolyze to 4-nitrophenol (hydrolysis product of parathion) in different soils under flooded conditions (Ferris and Lichtenstein, 1980 Sudhakar-Barik and Sethunathan, 1978). 

Smolen JM, Stone AT (1998) Organophosphorous ester hydrolysis catalyzed by dissolved metals and metal containing surfaces. In PM Huang et al. (eds) Soil chemistry and ecosystem health. Soil Sci Soc Am Spec Publ 52 Madison, Wisconsin, pp 157-171 Spencer WF, Shoup TD, Spear RC (1980) Conversion of parathion to paraoxon on soil dusts and clay-minerals as affected by ozone and UV-Ught. J Agric Food Chem 28 366-371 Stevenson FJ (1982) Humus chemistry. Wiley, New York Stevenson FJ (1986) Cycles of soil. Wiley Interscience, New York... 

The pesticides methyl and ethyl parathion were determined in run-off water er preconcentration on XAD-2. This allowed analyses of these compounds at the parts per billion level (497). Parathion and paraoxon obtained from leaf extracts and orchard soil have also been determined (492). The separation of 30 carbamate pesticides by RPC has been described (493). Various modes of postcolumn fluorometric detection of carbamate insecticides have been reported including post-colun)n reaction between o-phthalaldehyde and methylamine, a carbamate hydrolysis... 

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