BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on μ-Bondapak C-18 column. A molecular mass of 14358.69 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35–45°C, and required Ca2+. In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNFα were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively.

Snake venoms are important sources of substances with a variety of pharmacological activities. Among the different proteins present in these venoms, snake venom serine proteinases (SVSPs) have important effects on the hemostatic system that influence the hemodynamic properties of blood. Bothrops genus snakes presented their venom richly composed of SVSPs thrombin-like, and the isolation of these enzymes is of great interest.In 1994, the Center for the Study of Venoms and Venomous Animals (CEVAP) -UNESP standardized the fibrin sealant derived from snake venom, replacing the bovine thrombin by gyroxin thrombin-like enzyme from Crotalus durissus terrificus (Rattlesnake) and human plasma fibrinogen by buffaloes cryoprecipitate. Despite chromatographic techniques for the purification of gyroxin be well grounded in the literature, that income is considered low. Thus, in addition to gyroxin, other thrombin-like enzymes could be employed in the composition of the new fibrin sealant after being standardized to the purifying and chromatographic performance and widely evaluated for biological activities.Therefore, it is extremely important that in our lab is deployed, standardized and validated a method for the chromatographic purification of other thrombin-like enzymes such as found in Bothrops snake venoms. Thus a two-step chromatographic procedure was developed to routinely purify serine proteinases from Bothrops alternatus and B. moojeni snakes venoms to provide new enzymes for improving the CEVAP's heterologous fibrin sealant.

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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 αfibrinogenase 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.