AN AUTOMATABLE MICROASSAY FOR PHENYL VALERATE ESTERASE-ACTIVITIES SENSITIVE TO ORGANOPHOSPHORUS COMPOUNDS

An automatable microassay method developed for phenyl valerate esteras e (PVase) activity has been applied to determine the following activit ies in the soluble fraction of hen sciatic nerve: activity A (total PV ase activity), activity B (paraoxon-resistant PVase activity), activit y C {PVase activity resistant to 40 mu M paraoxon and 250 mu M mipafox } and neuropathy target esterase (NTE) activity (resistant to 40 mu M paraoxon but sensitive to 250 mu M 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 10 00 Station was used, which guarantees both inter- and intra-assay repr oducibility of the results, and shortens the total assay time. The tec hnical problems involved when processing many samples were thus resolv ed and with same regards it can also apply manually and using a microp late reader. In the case of activity A, the sensitivity of the method allowed the detection of activity in 1 mu g of protein (0.15 mg fresh sciatic nerve tissue), and the response was linear for different conce ntrations of 0.15-1.7 mg fresh tissue. For B, C and NTE, sensitivity c orresponded to 10 mu g of protein (1.5 mg fresh tissue in the microass ay), with a linear response in the range of 1.5-17 mg fresh tissue. Th e response was linear versus the time of enzyme-substrate reaction (30 -150 min). As tissue concentration increased, the response became nonl inear at shorter time. The procedure may be used to measure other enzy matic activities that yield phenols and chlorophenols as reaction prod ucts.