Abstract
Snake venoms are important sources of complex substances with a variety of pharmacological activities. Among them serine proteinases (SVSPs) have important effects on the hemostatic system influencing the hemodynamic of human or animal blood. Bothrops genus-snake venoms are rich in the thrombin-like enzyme, a type of SVSPs, with great interest to produce medicine. Therefore, the aim of this work was to describe a rapid, only two-step chromatographic-procedure developed to perform a faster purification of SVSPs from Bothrops alternatus and Bothrops moojeni venoms. As a result, two groups of serine proteinases respectively BaIII-4 - 8 and BmIII-2 - 5, were isolated and their molecular masses estimated by mass spectrometry and SDS-PAGE under denaturing conditions. The SVTLEs isolated from B. alternatus (BaIII-3 - 8) and B. moojeni (BmIII-2 - 5) fractions displayed apparent molecular mass around 30-40 kDa which closely relates to SVTLEs from other Bothrops species, as well their amino acid partial sequence triptych ions. Analysis of the alignment of the amino acid residue sequences of the N-terminal of the isolated proteins revealed a high level of identity with other SVTLEs. These enzymes coagulated plasma and showed fibrinogenolytic activity in blood. These SVTLEs isolated can be considered α-fibrinogenase mainly due to the fact that they hydrolyze the Aα chain fibrinogen. B. moojeni SVTLE showed greater activity than those from B. alternatus isolated. This new purification alternative approach developed was faster and more economical than the traditional process currently used. Faster purification and improved extraction yield can provide new insights into these enzymes including the use as a candidate molecule in the production of new drugs.
Highlights
Bothrops snake venoms contain a large variety of proteins and peptides affecting the hemostatic system; these proteins are classified as coagulant, anticoagulant or fibrinolytic factors [4, 44, 46]
The venoms were obtained from a venom-pool had been milked from Bothrops snakes (B. alternatus and B. moojeni) adults, of both sexes, individually microchipped, created and maintained in the CEVAPs Serpentarium, located at CEVAP-UNESP, Botucatu, São Paulo Brazil, according to the methodology developed by this Translational Center
The elution profiles of B. alternatus and B. moojeni venoms subjected to an affinity chromatography showed 3 fractions each (BaI-III andBmI-III, respectively) (Figure 1A, B)
Summary
Bothrops snake venoms contain a large variety of proteins and peptides affecting the hemostatic system; these proteins are classified as coagulant, anticoagulant or fibrinolytic factors [4, 44, 46]. A group of serine proteinases enzymes convert fibrinogen into fibrin by cleaving fibrinopeptides A and/or B. As this activity resembles the activity of thrombin, these venom components are commonly named “thrombin-like” enzymes (SVTLEs) [41, 43]. These proteins are good tools to study molecular details of activation of specific factors involved in coagulation and fibrinolytic cascades. They are useful in treating various thrombotic and hemostatic conditions after a medicine developed [9, 28]. In addition to affecting the hemostatic system these proteins affect platelet aggregation, American Journal of Biomedical and Life Sciences 2021; 9(4): 209-218 complement system, blood pressure and nervous system [23, 34]
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