Avaliação da atividade e da inibição de ureases: estudos biofísicos de interação e desenvolvimento de metodologias analíticas

Abstract

Urease catalyzes the hydrolysis of urea in NH3(g) and CO2(g); it is associated with problems in agriculture, such as reduced efficiency of urea fertilizer, and health, because it provides a microenvironment favorable to the colonization of the human stomach by H. pylori bacteria. Therefore, inhibiting and determining urease activity in different matrices is important. Thus, the main objective is to evaluate the mechanism of urease inhibition by organic compounds (natural and synthetic) using biophysical studies, as well as the development of methodologies to determine the activity of ureases in soils and saliva (synthetic and human), in addition to evaluating potential inhibitors. For the interaction between urease and natural isothiocyanate (MFC), an IC50 = 487 µM was obtained, and the compound was classified as a mixed inhibitor. The synchronized fluorescence assays corroborated the nature of the inhibition process. Spectroscopic studies indicated conformational changes in the enzyme resulting from the MFCurease interaction, with a binding constant (Kb) equal to 1.80 × 102 L mol-1 and stoichiometry 1: 1. In addition, the formation of the corresponding dithiocarbamates and thioureas has been proven after the reaction of the MFC with amino acids containing thiol and amine/guanidine groups. Thus, the urease's inhibition mechanism by MFC occurred through covalent and noncovalent interactions. MFC was more effective in soil microbiota than in vitro, with inhibition equivalent to NBPT for the two soils evaluated. For the interaction of urease with five benzoylthioureas, a direct relationship was found with the size of the N-alkyl substituent with the IC50 (M) and Kb values. It was found that BTU1 was the most active and, therefore, selected for biophysical studies in soils with urease. For the BTU1-urease complex, Kb was on the order of 103 L mol-1 at the different temperatures evaluated; based on thermodynamic studies, the preferred forces for stabilizing the supramolecular complex were hydrogen bonds and van der Waal forces. Synchronized fluorescence and competition assay studies indicate that BTU1 probably has classic mixed inhibitor behavior. In the different soils evaluated, BTU1 showed an inhibition potential equivalent to NBPT, emphasizing the importance of in vitro assays and application in a real sample for selecting a potential inhibitor. Two analytical paper devices (UrePAD and Multicolor PAD) were developed to determine urease activity in soils, saliva, and in the absence/presence of inhibitors. UrePAD is based on the color change of the acid-base indicator phenol red, exploring the change in pH due to the hydrolysis of urea by urease. The Multicolor PAD is also a colorimetric device employing five acid-base indicators (phenol red, bromothymol blue, bromocresol purple, m-cresol purple, and neutral red) and surface-modified polyelectrolyte poly-4-styrene sulfonate paper. sodium or polyvinylpyrrolidone. The images obtained were digitalized in a bench scanner, and the analysis was performed in the Corel Draw X8 software. The methods showed a mean LOD of 0.10 U mL-1 with linearity between 0.25 and 4.0 U mL-1 . Both devices were applied to determine urease activity in four soil samples with different characteristics and in vitro evaluation of classic inhibitors. The results obtained did not differ statistically (95% confidence) from the reference method of indophenol blue, having as advantages the low cost, reduced volume of reagents/sample combined with simplicity and portability, showing the ability to simplify the determination of the activity of the urease in soils and the evaluation of potential inhibitors. Finally, the Multicolor PAD was applied to determine urease in a sample of synthetic and human saliva, with quantitative recoveries (between 80 - 120%) and to show a more significant variation in color intensity for a human saliva sample from an individual infected with H. pylori. Thus, it is possible to use a simple, fast, low-cost method to diagnose infection by ureolytic bacteria such as H. pylori.