An automatable microassay for phenyl valerate esterase activities sensitive to organophosphorus compounds.
An automatable microassay method developed for phenyl valerate esterase (PVase) activity has been applied to determine the following activities in the soluble fraction of hen sciatic nerve: activity A (total PVase activity), activity B (paraoxon-resistant PVase activity), activity C (PVase activity resistant to 40 microM paraoxon and 250 microM mipafox) and neuropathy target esterase (NTE) activity (resistant to 40 microM paraoxon but sensitive to 250 microM mipafox), operationally defined as activity (B-C). This microassay is based on the technique described by Barril et al. (Toxicology. 1988. 49:107-114). The Automated Biomek 1000 Station was used, which guarantees both inter- and intra-assay reproducibility of the results, and shortens the total assay time. The technical problems involved when processing many samples were thus resolved and with same regards it can also apply manually and using a microplate reader. In the case of activity A, the sensitivity of the method allowed the detection of activity in 1 microgram of protein (0.15 mg fresh sciatic nerve tissue), and the response was linear for different concentrations of 0.15-1.7 mg fresh tissue. For B, C and NTE, sensitivity corresponded to 10 micrograms of protein (1.5 mg fresh tissue in the microassay), with a linear response in the range of 1.5-17 mg fresh tissue. The response was linear versus the time of enzyme-substrate reaction (30-150 min). As tissue concentration increased, the response became nonlinear at shorter time. The procedure may be used to measure other enzymatic activities that yield phenols and chlorophenols as reaction products.
Escudero
M A
MA
Department of Neurochemistry, Alicante University, Spain.
Sogorb
M A
MA
Vilanova
E
E
D000818:Animals; D001331:Automation / Q000379:methods*; D015415:Biomarkers / Q000032:analysis*; D002265:Carboxylic Ester Hydrolases / Q000032:analysis*; D005260:Female; D008832:Microchemistry / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015415:Biomarkers; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase