Estevez_2004_Toxicol.Lett_151_171

In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act=A1e-k1It+A2e-k2It+AR (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as AR=15.1% (step 2). The model equation with a fixed AR value was again applied (step 3) to deduce the second-order inhibition rate constants (k1=2.6 x 10(6) M-1 min-1 and k2=0.28 x 10(6) M-1 min-1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of AR, k1, and k2, the amplitudes for the two exponential (sensitive) components (A1 and A2) were re-estimated (A1=50.2% and A2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 microM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.