Simultaneous enzymatic kinetic determination of pesticides, carbaryl and phoxim, with the aid of chemometrics.

A sensitive and selective enzymatic kinetic method for the simultaneous determination of mixtures of carbaryl and phoxim pesticides was researched and developed. It was based on the inhibitory effect of the pesticides on acetylcholinesterase (AChE), and the use of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as a chromogenic reagent for the thiocholine iodide (TChI) released from the acetylthiocholine iodide (ATChI) substrate. The DTNB-thiocholine reaction was investigated by a spectrophotometric-kinetic approach. The complex rate equation for the formation of the chromogenic product, P, was solved under certain experimental conditions, which enabled the absorbance (A(P), at lambda(max)=412 nm) from the mixtures of the two pesticide inhibitors to be directly related to their concentrations provided the absorbance additivity was followed. The spectra were measured for mixtures of carbaryl and phoxim at different concentrations, and at t=904 s, T=35 degrees C, pH=7.5, c(ATChI)=0.14, and c(AChE)=0.10 mg mL(-1). The detection limits of the enzymatic kinetic spectrophotometric procedures for the determination of the carbaryl and phoxim were 4.7 and 0.59 microg L(-1), respectively. Calibration models for chemometrics methods, such as principal component regression (PCR), partial least squares (PLS) and radial basis function-artificial neural network (RBF-ANN) were constructed and verified with synthetic samples of the mixtures of the two pesticides. The best performing model was based on the RBF-ANN method yielding at approximately 10 ppb analyte concentrations, %RPE(T) (carbaryl=5.2; phoxim=6.5), %Recovery (approx.105%) and %RPE(T) (6.5). Various spiked town-water samples produced recoveries in the range of 98.8-103% for each pesticide.

Kinetic mechanism of choline kinase from rat striata.

The kinetic mechanism of choline kinase associated with both the cytosolic and membrane fractions of synaptosomes isolated from rat striata was studied. The velocity of choline kinase was measured using various concentrations of MgATP at several concentrations of uncomplexed Mg2+ and a single concentration of choline. This experiment was repeated using different concentrations of choline. Analysis of these data according to a terreactant mechanism indicates that MgATP binds in rapid equilibrium prior to Mg2+, but the binding of MgATP and choline is random. Product inhibition by phosphorylcholine was noncompetitive versus both choline and MgATP. Hemicholinium-3 (HC-3), an analog of choline and competitive inhibitor of the sodium-dependent high affinity choline transport system, was noncompetitive versus choline and uncompetitive versus MgATP at high levels of Mg2+. However, when the concentration of Mg2+ was decreased below the KMg2 +, HC-3 was noncompetitive versus MgATP. Thiocholine, another analog of choline, gave slope-linear intercept hyperbolic inhibition versus choline. Mg-5'-adenylyl imidodiphosphate, an analog of MgATP, was competitive versus MgATP and noncompetitive versus choline. Virtually identical results were obtained using either soluble or particulate forms of choline kinase from rat striata. All data are consistent with the mechanism suggested by initial velocity studies alone and additionally suggest that the release of MgADP is slow, occurs last, and may limit the overall rate of the reaction.