ИСТИНА

The ability of organophosphates (OP) to effectively inhibit cholinesterases is exploited in the most of the sensitive methods of the OP detection in the environment. The aim of this work was to develop a new biosensor for the analysis of butyrylcholinesterase (BChE) and its inhibitors. This method is based on the combination of BChE enzymatic hydrolysis of new substrate - phenyl valerate with amperometric phenol detection by the Clark-rype oxygen electrode modified by immobilized tyrosinase. The determination of the amount of phenol released was carried out in a kinetic regime. The biosensor developed is characterized by a wide range of linearity with respect to phenol (from 5E-07 up to 1E-04 M) and an excellent operational stability during 3 weeks at room temperature. The detection limit for BChE was found to be 1.5E-04 M. The equilibrium (KM) and kinetic (kcat) parameters of phenyl valerate hydrolysis in the presence of horse serum BChE were determined by means of tyrosinase biosensor at pH 7.0 and were found to be 0.2 mM and 177 c-1. The number of active sites used for the calculation of kcat was found by means of both the amperometric detection with phenyl valerate and the spectrophotometric detection with butyrylthiocholine from the data of BChE titration by diisopropylfluorophosphate. The number of active sites was calculated to be 35% in each case. The influence of pH in the range of 4.5-9.0 on the kinetic parameters of phenyl valerate hydrolysis was investigated and the optimal pH conditions of BChE activity analysis were determined with a use of biosensor techniques. We think our proposal to use such an approach for the BChE detection is promising owing to great progress in the phenol measuring sensors.