Browsing by Author "Fernandes, Pedro Daniel de Jesus Conde"
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- Physical and Chemical Parameters Screening for Co-Crystallization of rS -COMT-entacaponePublication . Fernandes, Pedro Daniel de Jesus Conde; Castro, Filipa Juliana Fernandes; Passarinha, Luís António PaulinoCatechol-O-methyltransferase (COMT, EC 2.2.1.6) is the enzyme responsible for the Omethylation of substrates such as catecholamines and estrogens with a typically catechollic structure. Commonly, COMT can be stabilized through specific molecules capable of increasing its thermodynamic stability, thus preventing its denaturation and loss of activity. After ensuring optimal conditions for protein activity, stability, and purity, carrying out structural studies is the next step. In these studies, crystallization tests are carried out with the aim of forming a crystal, in which the protein molecules are organized in a regular and orderly manner, to obtain information about their three-dimensional structure. In this work, we evaluated the effect of the interaction of ionic liquids on the enzymatic activity of SCOMT and investigated the crystallization conditions of purified SCOMT fractions. Firstly, we studied the influence of two ionic liquids, choline dihydrogen phosphate ([Ch][DHP]) and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), on the SCOMT lysate for 12 hours by applying a storage temperature of 4 ºC or -80 ºC and whether the ionic liquids would increase the stability of the protein, evaluating its specific activity. A greater potential stabilizing effect was obtained using [C4mim]Cl at 4 °C, with an activity recovery of about 350% over 12 hours. Then, crystallization tests of purified SCOMT fractions were carried out, using the vapor diffusion (Hanging Drop) and counter diffusion methods, where several factors were evaluated, namely, temperature (4 ºC and 20 ºC), concentration and composition of the precipitating agent solution (HEPES, pH=7.5, methyl-2,4-pentanediol and Caps, pH 10.5, Li2SO4, NaH2PO4/ K2HPO4) and the influence of S-adenosyl-L-methionine (SAM) and 3.5 -dinitrocatechol (DNC), a cofactor and a COMT inhibitor, respectively. By evaluating the various factors, we were able to conclude that better results were obtained at a temperature of 20 ºC, which corresponds to a precipitating agent solution composed of Caps, pH 10.5, Li2SO4, NaH2PO4/K2HPO4 and that the steam diffusion technique showed more promising results. In addition, a high concentration of precipitates was observed throughout the tests, mostly DNC and, in the minority, salts present in the solution of the precipitating agent. In a last step, the results obtained were classified, namely, distinguishing protein crystals from salt crystals, but due to their small size, X-ray identification was not possible. Thus, as an alternative, tests such as the absorption of dyes (Timol Blue and Coomassie Blue) were carried out and the Crush Test. These tests showed inconclusive results, since, due to the small size of the crystals, it was not possible to identify their nature with certainty.