- Email: [email protected]
23.P4. Partial purification of prothrombin from Bothrops jararaca plasma
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Abstracts / Comparative Biochemistry and Physiology, Part A 148 (2007) S103–S108
and was able to preferentially hydrolyze the Aα-chain of fibrinogen and the α-chain of fibrin. Its activity was inhibited by metal-chelating agents. In addition, its enzymatic activity was stimulated by the divalent cations Ca2+ and Mg2+ but inhibited by Zn2+ and Cu2+. Hemorrhage induced by leuc-B (MHD = 30 ng in rabbit) should be the result of the degradation of extracellular matrix and sub-endothelium components leading to the disruption of blood vessel, with alterations in platelet function. Support: FAPEMIG, FUNED,CNPq and Fundação Araucaria. doi:10.1016/j.cbpa.2007.06.275
23.P3. The coagulant effect of different snake venoms on purified Bothrops jararaca (Bj) fibrinogen Vieira, C.O., Sano-Martins, I.S., and Tanaka-Azevedo, A.M. Laboratório de Fisiopatologia, Instituto Butantan, Brazil
between 1 and 2 mM (Degen et al., 1983). Prothrombin is the precursor of the enzyme thrombin, an enzyme that plays an extraordinarily prominent role in the blood coagulation and wound healing mechanisms. The aim of this study is to purify Bothrops jararaca (B. jararaca) prothrombin and to compare it with human and other animals' prothrombin. This protein was obtained through barium chloride precipitation, two sequential steps of ammonium sulfate fractionation (35% and 70% saturation) and DEAE–cellulose chromatography. Our SDS– PAGE results suggest that the molecular mass of B. jararaca prothrombin is 80 kDa, which can be compared to human prothrombin, which has a molecular mass of 73 kDa, including its proteolytic coagulant activity. The perspectives for this work are to improve the purification process in order to get higherpurity protein and to compare biologically and biochemically with other animals' prothrombin. Financial support: CAPES and FAPESP (04/02224-4). doi:10.1016/j.cbpa.2007.06.277
[email protected] Most Brazilian poisonous snakes present in their venom a thrombin-like activity, i.e. an enzyme similar to thrombin which cleaves fibrinogen-generating fibrin. On the other hand, substances such as anticoagulant and antihemorrhagic activity have been described in the plasma. The aim of this work was to isolate Bj fibrinogen and to study the effect of different snake venom (Bothrops and Crotalus) on it. The Bj fibrinogen was obtained from plasma through barium chloride adsorption, ammonium sulfate precipitation and gel filtration chromatography. The molecular masses of Bj fibrinogen chains were 71, 60 and 55 for Aα, Bβ and γ, respectively, by SDS–PAGE. Minimum coagulant dose of Crotalus durissus terrificus (21.5 μg mL−1) and Bothrops jararaca (2 μg mL−1) venoms were used for clotting time tests. Bj fibrinogen did not clot or had prolonged coagulation time with Bothrops venoms used; however, it was coagulated by C. durissus terrificus venom. These results indicate that Bj fibrinogen was adapted to protect these animals against their own venom coagulant activity. Supported by FAPESP (04/02224-4) and CNPq. doi:10.1016/j.cbpa.2007.06.276
23.P4. Partial purification of prothrombin from Bothrops jararaca plasma Morais, K.B., Sano-Martins, I.S., and Tanaka-Azevedo, A.M. Laboratório de Fisiopatologia, Instituto Butantan, Brazil [email protected] Prothrombin is the most abundant of the vitamin K-dependent blood clotting proteins, circulating at plasma concentrations
23.P5. Analysis of Bothrops jararaca coagulation inhibitor (BjI) similar proteins in Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrific Morais, K.B., Grego, K.F., and Tanaka-Azevedo, A.M. Laboratório de Fisiopatologia, Instituto Butantan, Brazil [email protected] Bothrops jararaca coagulation inhibitor (BjI) is a highmolecular weight protein isolated from B. jararaca snake blood which inhibits the thrombin coagulant activity (TanakaAzevedo et al., 2004). The aim of this work was to compare the anticoagulant effects of Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus snakes' plasmas and also to investigate the presence of BjI similar proteins in these species' blood. Thrombin times of snake plasmas were performed and their proteins were analyzed by SDS–PAGE. The antiserum containing anti-BjI obtained from mice was used in order to confirm if the prolonged thrombin times found in these snake plasmas were caused by the presence of immunological protein similar to BjI by Western blotting tests. As the same as B. jararaca plasma, the poisonous snakes plasmas also presented a prolonged thrombin time (31.6 s, 44.1 s and 45.4 s for B. alternatus, B. jararacussu and C. durissus terrificus, respectively) when compared to control (10 s). The snakes' plasma proteins analyzed by SDS–PAGE showed similar profiles. Our current results confirmed the presence of BjI similar proteins in B. alternatus, B. jararacussu and C. durissus terrificus plasmas by Western blotting. Financial support: FAPESP (nos. 04/ 02224-4 and 04/13434-0). doi:10.1016/j.cbpa.2007.06.278
Abstracts / Comparative Biochemistry and Physiology, Part A 148 (2007) S103–S108
and was able to preferentially hydrolyze the Aα-chain of fibrinogen and the α-chain of fibrin. Its activity was inhibited by metal-chelating agents. In addition, its enzymatic activity was stimulated by the divalent cations Ca2+ and Mg2+ but inhibited by Zn2+ and Cu2+. Hemorrhage induced by leuc-B (MHD = 30 ng in rabbit) should be the result of the degradation of extracellular matrix and sub-endothelium components leading to the disruption of blood vessel, with alterations in platelet function. Support: FAPEMIG, FUNED,CNPq and Fundação Araucaria. doi:10.1016/j.cbpa.2007.06.275
23.P3. The coagulant effect of different snake venoms on purified Bothrops jararaca (Bj) fibrinogen Vieira, C.O., Sano-Martins, I.S., and Tanaka-Azevedo, A.M. Laboratório de Fisiopatologia, Instituto Butantan, Brazil
between 1 and 2 mM (Degen et al., 1983). Prothrombin is the precursor of the enzyme thrombin, an enzyme that plays an extraordinarily prominent role in the blood coagulation and wound healing mechanisms. The aim of this study is to purify Bothrops jararaca (B. jararaca) prothrombin and to compare it with human and other animals' prothrombin. This protein was obtained through barium chloride precipitation, two sequential steps of ammonium sulfate fractionation (35% and 70% saturation) and DEAE–cellulose chromatography. Our SDS– PAGE results suggest that the molecular mass of B. jararaca prothrombin is 80 kDa, which can be compared to human prothrombin, which has a molecular mass of 73 kDa, including its proteolytic coagulant activity. The perspectives for this work are to improve the purification process in order to get higherpurity protein and to compare biologically and biochemically with other animals' prothrombin. Financial support: CAPES and FAPESP (04/02224-4). doi:10.1016/j.cbpa.2007.06.277
[email protected] Most Brazilian poisonous snakes present in their venom a thrombin-like activity, i.e. an enzyme similar to thrombin which cleaves fibrinogen-generating fibrin. On the other hand, substances such as anticoagulant and antihemorrhagic activity have been described in the plasma. The aim of this work was to isolate Bj fibrinogen and to study the effect of different snake venom (Bothrops and Crotalus) on it. The Bj fibrinogen was obtained from plasma through barium chloride adsorption, ammonium sulfate precipitation and gel filtration chromatography. The molecular masses of Bj fibrinogen chains were 71, 60 and 55 for Aα, Bβ and γ, respectively, by SDS–PAGE. Minimum coagulant dose of Crotalus durissus terrificus (21.5 μg mL−1) and Bothrops jararaca (2 μg mL−1) venoms were used for clotting time tests. Bj fibrinogen did not clot or had prolonged coagulation time with Bothrops venoms used; however, it was coagulated by C. durissus terrificus venom. These results indicate that Bj fibrinogen was adapted to protect these animals against their own venom coagulant activity. Supported by FAPESP (04/02224-4) and CNPq. doi:10.1016/j.cbpa.2007.06.276
23.P4. Partial purification of prothrombin from Bothrops jararaca plasma Morais, K.B., Sano-Martins, I.S., and Tanaka-Azevedo, A.M. Laboratório de Fisiopatologia, Instituto Butantan, Brazil [email protected] Prothrombin is the most abundant of the vitamin K-dependent blood clotting proteins, circulating at plasma concentrations
23.P5. Analysis of Bothrops jararaca coagulation inhibitor (BjI) similar proteins in Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrific Morais, K.B., Grego, K.F., and Tanaka-Azevedo, A.M. Laboratório de Fisiopatologia, Instituto Butantan, Brazil [email protected] Bothrops jararaca coagulation inhibitor (BjI) is a highmolecular weight protein isolated from B. jararaca snake blood which inhibits the thrombin coagulant activity (TanakaAzevedo et al., 2004). The aim of this work was to compare the anticoagulant effects of Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus snakes' plasmas and also to investigate the presence of BjI similar proteins in these species' blood. Thrombin times of snake plasmas were performed and their proteins were analyzed by SDS–PAGE. The antiserum containing anti-BjI obtained from mice was used in order to confirm if the prolonged thrombin times found in these snake plasmas were caused by the presence of immunological protein similar to BjI by Western blotting tests. As the same as B. jararaca plasma, the poisonous snakes plasmas also presented a prolonged thrombin time (31.6 s, 44.1 s and 45.4 s for B. alternatus, B. jararacussu and C. durissus terrificus, respectively) when compared to control (10 s). The snakes' plasma proteins analyzed by SDS–PAGE showed similar profiles. Our current results confirmed the presence of BjI similar proteins in B. alternatus, B. jararacussu and C. durissus terrificus plasmas by Western blotting. Financial support: FAPESP (nos. 04/ 02224-4 and 04/13434-0). doi:10.1016/j.cbpa.2007.06.278