Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis
Introduction
The vast majority of snakebites in Latin America are induced by species of the genus Bothrops (Fan and Cardoso, 1995, Gutiérrez and Lomonte, 1995). These envenomations are characterized by a complex pathophysiology which includes rapid and drastic local tissue damage and systemic manifestations (França and Málaque, 2003). Serine proteases are common constituents of venom proteomes and venom gland trancriptomes of viperid species (Francischetti et al., 2004, Kashima et al., 2004, Cidade et al., 2006, Serrano and Maroun, 2005).
Abundant serine proteases have been isolated and characterized from snake venoms (Serrano and Maroun, 2005). Despite sharing similar structural features, venom serine proteases display a highly diverse pharmacological profile. This includes actions on proteins of the coagulation cascade, such as thrombin-like activity on fibrinogen, activation of factor V, activation of protein C, fibrinogenolysis, activation of plasminogen, and induction of platelet aggregation (Markland, 1998, Serrano and Maroun, 2005).
Thrombin-like enzymes from snake venoms belong to a class of serine proteases that can cause blood clotting in vitro, a feature exhibited by several snake venoms (Pirkle, 1998, Magalhães et al., 2003). These enzymes render the blood uncoagulable when acting in vivo apparently by depleting the circulating fibrinogen. Parenteral administration of TLEs to human subjects (Bell, 1988, Hessel and Blombäck, 1971) or to experimental animals (Damus et al., 1972, Silberman et al., 1973) produces an abrupt fall in the plasma concentration of fibrinogen, with little or no effects on other hemostatic factors. Some of these thrombin-like enzymes from snake venoms have been used as anticoagulant agents for the prevention and treatment of a wide range of thrombotic disorders, for organ transplants, vascular surgery and are widely used in laboratories in the detection of fibrinogen in samples of heparinized blood and as a reagent in coagulation studies (Marsh and Williams, 2005). Although the list of biochemical and biological actions attributed to thrombin continues to grow, the term thrombin-like as applied to venom enzymes generally implies only the capacity to induce the clotting of fibrinogen. During the last phase of the coagulation process, thrombin promotes the formation and stabilization of the fibrin gel by limited proteolysis of two distinct plasma proteins: fibrinogen and coagulation factor XIII.
In Brazil, snakes of the genus Bothrops (lanceheads) are responsible for 90% of all recorded snakebites in which the snake is identified. Bothrops marajoensis is a serpent found at the island of Marajó, North of Brazil. The venom of the gender Bothrops induces local effects characterized by hemorrhage, necrosis, edema and intense pain. In spite of its importance, clinical studies on the mechanisms involved in the genesis of these phenomena are still scarce. Therefore, it was considered of interest to purify the serine protease with thrombin-like activity enzyme from B. marajoensis venom and to elucidate some of its physicochemical, structural and functional properties. In the present communication, we describe a one step purification procedure and partial characterization of a fibrinogen-clotting enzyme from B. marajoensis venom, hereafter referred to as TLBm. We also present the complete amino acid sequence of TLBm and discussed its homology with other thrombin-like snake venom.
Section snippets
Reverse phase HPLC
TLBm from B. marajoensis venom was purified by reverse phase HPLC according to the method described by Ponce-Soto et al. (2007). Briefly, 5 mg of the whole venom was dissolved in 250 μl of buffer A (0.1% trifluoroacetic acid–TFA) and centrifuged at 4500 g and the supernatant was then applied on the analytical reverse phase HPLC μ-Bondapak C18, previously equilibrated with buffer A for 15 min. The elution of the protein was then conducted using a linear gradient of buffer B (66.5% Acetronitrile in
Results
Fractionation of B. marajoensis venom by reverse phase HPLC (Fig. 1) showed the elution of eighteen main fractions (1–18). For this chromatographic procedure, protein concentration was monitored by measuring the absorbance at 280 nm. Proteolityc activity on the substrate DL-BAρNA was also determined and identified as thrombin-like activity in fraction 15 (Fig. 1). The degree of purity of this fraction was confirmed by analytical reverse phase HPLC, which indicated only one main fraction, and
Discussion
This article reports an efficient and relatively simple procedure for the isolation of a highly purified thrombin-like serine protease from B. marajoensis venom, which was named TLBm.
Our results using a simple and rapid procedure based on RP-HPLC showed that the B. marajoensis venom can be separated in eighteen fractions. Fraction fifteen presents thrombin-like activity and was denominated TLBm. To confirm the homogeneity, fraction fifteen was further purified using the same chromatographic
Acknowledgement
The authors thank Paulo A. Baldasso for technical assistance. This work was supported by the São Paulo State Research Foundation, (FAPESP) and is part of Ms Sc thesis of Augusto Vilca Quispe (Process 06/54275-7).
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