Identificação e separação de fosfolipases da peçonha de Bothrops alternatus com potencial antiparasitário e antitumoral

Abstract

Introduction: Snake venoms are complex mixtures of molecules with diverse actions. Its composition has compounds such as proteins, enzymes, peptides and other organic and inorganic compounds. Species of the genus Bothrops are responsible for most of the snakebites recorded and have an arsenal of biotoxins, such as phospholipases, which are low molecular weight proteins, characterized by their enzymatic action on membrane phospholipids. Research into therapeutic compounds of natural origin has been drawing attention in recent years, highlighting the need for studies to isolate snakebite toxins and prospect for new drugs. Objective: To identify and isolate proteins with antiparasitic and/or anticancer potential from the venom of the snake Bothrops alternatus. Methods: To identify proteins with therapeutic potential of B. alternatus, a bibliographic search was carried out in PubMed and CAPES databases, following the PRISMA method. Fractionation of the crude venom and purification of proteins of interest by liquid chromatography were performed. The molecular mass of the isolated proteins was analyzed by polyacrylamide gel electrophoresis under denaturing conditions. The phospholipase activity was carried out through a hydrolysis assay of phospholipids present in the chicken egg yolk. Results: The database search resulted in 210 articles, of which 15 articles were analyzed in detail. Screening resulted in 7 proteins with antitumor potential and 3 had proteins with antiparasitic, antiviral or antimicrobial potential in the venom of B. alternatus. The fractionation of the crude venom resulted in 6 main fractions, A1-A6, and in fractions A4 and A5 it was possible to identify the presence of proteins with molecular weight less than 30 kDa and with phospholipase activity, indicating the presence of PLA2. Based on these results, these fractions were submitted to hydrophobic interaction chromatography. The chromatography of the A4 fraction resulted in 3 subfractions, A4a-A4c and showed separation of low molecular weight proteins in the A4c subfraction, but with low protein yield. Chromatography of the A5 fraction resulted in 7 subfractions, A5a-A5g, and protein components with similar characteristics to BaltPLA2 phospholipase, a phospholipase of B. alternatus with antiviral potential against dengue, were identified in the A5g subfraction. Conclusions: The venom of the B. alternatus snake contains several bioactive components, especially enzymes with antiparasitic, antimicrobial and/or antitumor effects. The purification methods developed in this work allowed the obtaining of relevant fractions with enzymatic activity, but with low protein yield. More refined methodologies should be employed in order to obtain a purification with a higher yield and degree of purity, indicating that further studies are needed.