Estudo microcalorimétrico da inibição da acetilcolinesterase pelo agrotóxico carbaryl

Abstract

Carbaryl is a carbamate insecticide and a competitive inhibitor of the enzyme acetylcholinesterase, an enzyme responsible for the hydrolysis of acetylcholine and subject of several studies in different fields of science. Most of the techniques used to screen enzyme kinetics employ modified or synthetic substrates capable of generating a chemical or an electrical signal, recognizable by some equipment. For studies using acetylcholinesterase, acetylthiocholine is used as a substrate, where one of its catalytic products, the thiocholine, is capable of reacting with some molecules having the property of absorbing light at one wavelength. This coupled reaction can vary according to the experimental conditions and alter the result of the aforementioned reaction, namely the acetylthiocholine hydrolysis. This method is not able to inform about the hydrolysis of acetylcholine. Direct techniques may be used, but are more sophisticated, an example is isothermal titration calorimetry. The technique can show not only the hydrolysis reaction of acetylthiocholine but also of acetylcholine, through the heat exchange that occurs during catalysis, not requiring any other molecules, but the enzyme and its substrate only. In order to compare the kinetic values obtained in the hydrolysis of both substrates; this work has employed two techniques, spectrophotometric and calorimetry. The spectrophotometric technique created by Ellman uses a molecule as chromophore agent. The calorimetric technique can create conditions where the substrate can accumulate in the reaction cell and the kinetics is determined by the amount of heat exchanged by time. Different mathematical analysis, common in enzymology area, was used to compare the results obtained in the reactions using acetylcholine and acetylthiocholine, at the two methods employed. The present study utilized different concentrations of carbaryl to determine the inhibition constant thereof also by spectrophotometric and calorimetric methods, using different mathematical analysis. The results show a similarity between the values obtained with both substrates, these being considerably greater for acetylcholine, the natural substrate of the enzyme. Moreover, both techniques, although with larger errors in spectrophotometric, were similar to each other. This shows the robustness of the calorimetric technique in the kinetic study, and the proximity of acetylthiocholine values with acetylcholine shows that the use of this synthetic substrate returns values similar to the natural substrate.