Abstract
BackgroundSnake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA2) from Bothrops atrox venom, and biochemically characterize these molecules to enable future functional studies.MethodsTo obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.ResultsThe metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA2.ConclusionsThe present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.
Highlights
Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides
The interest in the biochemical and functional characterization of toxins isolated from snake venoms is due to their relevance in envenomations, and to their potential use as valuable research tools in different areas of knowledge
Numerous attempts have been made to use these compounds as tools for research and for applications in the medical field, and as such, the purification and characterization of snake toxins are of utmost importance
Summary
Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. The protein components include enzymes such as phospholipases A2 (PLA2s), Lamino acid oxidases (LAAOs), serine proteases (SVSPs) and metalloproteases (SVMPs) [6, 7]. These toxins and other components of snake venoms can act independently or synergistically to cause local or systemic tissue damage and various other toxic effects [8, 9]. In order to isolate specific proteins from snake venoms, which are highly complex and may present more than 100 protein components [10], usually two or more chromatographic steps are needed, which may include steps of molecular exclusion, ion exchange, affinity, reverse phase, among others. The choice of chromatography type depends on the specific characteristics of each protein to be isolated
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