Paraoxon measuring hydrolysis

Human serum paraoxonase (PON 1) is an esterase that is physically associated with high-density lipoprotein (HDL) and is also distributed in tissues such as liver, kidney, and intestine [38,39]. Activities of PON 1, which are routinely measured, include hydrolysis of organophosphates, such as paraoxon (the active metabolite of the insecticide parathion) hydrolysis of arylesters, such as phenyl acetate and lactonase activities. Human serum paraoxonase activity has been shown to be inversely related to the risk of cardiovascular disease [40,41], as shown in atherosclerotic, hypercholester-olemic, and diabetic patients [42-44]. In 1998 HDL-associated PON 1 was shown to protect LDL, as well as the HDL particle itself, against oxidation induced by either copper ions or free radical generators [45,46], and this effect could be related to the hydrolysis of the specific lipoproteins oxidized lipids such as cholesteryl linoleate hydroperoxides and oxidized phospholipids. Protection of HDL from oxidation by PON 1 was shown to preserve... 

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. 

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... 

Paraoxonase/arylesterase from human serum and other manunalian species catalyzes the hydrolysis of organophosphates, aromatic carboxylic acids and possibly carbamates (Primo-Parmo et al. 1996). Paraoxonase (PONl) is so called because paraoxon is the substrate commonly used to measure its enzyme activity (Mackness et al. 1998). The crystal structure of PONl family of calcium-containing... 

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