Biochemical, structural and functional evaluation of a phosphodiesterase from Crotalus durissus collilineatus venom

Abstract

Snake venoms are represented by a wide range of components able to induce various pathological symptoms at envenoming victims, which is part of the neglected circumstances by WHO and is also a serious public health problem in Brazil. In the national territory, the genus Crotalus is represented by the snakes of species Crotalus durissus, scattered in several regions. Their venoms present a great complexity of protein components, being able to influence in the toxicity of the crotalic accident, hindering their treatment. In order to elucidate the toxicity mechanism of the various components is necessary the molecule isolation and its characterization. Thus, its action at envenoming becomes known, which facilitates the treatment of symptoms. Due to the complexity of C. d. collilineatus venom, many components have not yet been isolated, as phosphodiesterase (PDE). These enzymes are part of the nucleases, with DNAse and RNAse, and have a high molecular weight (90 ~160 kDa), and they can be found in monomeric or dimeric form. PDEs can be classified as 1 to 11 and they are responsible for breaking of phosphodiester bonds of nucleic acids, ATP, ADP, NAD, NGD, cAMP and cGMP, being able to interfere in physiological or pathological processes, which make them therapeutic targets of various diseases, besides acting as molecular tools. Their role in the snakebite envenoming is still not fully elucidated, but it is known that PDE is capable to leading to hypotension and locomotor depression of the prey and also capable of inhibiting platelet aggregation in vitro. Therefore, the aim of this study will perform the isolation of PDE from C. d. collilineatus venom, through the combination of liquid chromatography techniques, its identification by mass spectrometry techniques and amino-terminal sequencing, the determination of its molecular mass, build its three-dimensional model, evaluate its stability at different pH and temperature conditions, as well as to evaluate the phosphodiesterase activity on the bis(p-nitrophenyl)-phosphate substrate at different pH and temperature conditions and determine its kinetic parameters on the same substrate, evaluate the specificity of inhibition of the enzyme by 5 different inhibitors, verify that the antivenom produced in Brazil is able to recognize the PDE through indirect ELISA and perform platelet aggregation assay with this enzyme. In this way, the present study becomes relevant because will analyze a toxin that is important for the snakebite envenoming, but still little studied, increasing the knowledge about this enzymatic class. (AU)