The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some bi... more The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.
The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some bi... more The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.
Toxicon : official journal of the International Society on Toxinology, 2012
This communication describes the general biochemical properties and some immunological characteri... more This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD 50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.
Las serpientes Bothrops sp. causan el mayor número de casos de ofidismo en el Perú, su veneno con... more Las serpientes Bothrops sp. causan el mayor número de casos de ofidismo en el Perú, su veneno contiene enzimas que participan en la difusión de la ponzoña, así como en sus efectos miotóxicos, edemáticos y de alteración en la agregación plaquetaria. Objetivos. Evaluar el efecto del antiveneno botrópico polivalente al estado líquido producido por el Instituto Nacional de Salud (INS) sobre la fosfolipasa A 2 (PLA2), L-aminoácido oxidasa (LAO) y hialuronidasa (HA) de los venenos de B. atrox, B. barnetti, B. brazili y B. pictus. Materiales y métodos. La PLA2 fue determinada por el retardo en el tiempo de coagulación de una emulsión lipoproteica al 45%, LAO usando Lleucina como substrato en presencia de O-dianisidina y HA empleando ácido hialurónico y el reactivo turbidimétrico BCTA, se usó para cada enzima ½, 1 y 2 dosis del antiveneno al estado natural o calentado a 37 °C durante cinco días ensayados por triplicado. Resultados. HA fue la enzima más neutralizada por el antiveneno, todos los venenos con excepción de B. brazili fueron totalmente inhibidos a cualquier dosis. Para LAO se tuvieron valores de inhibición de 68 a 100% usando dos dosis del antiveneno, mientras que PLA2 fue la menos inhibida (70 a 80%) a dos dosis. Con el antiveneno calentado se registró una disminución del efecto inhibitorio encontrado inicialmente. Conclusiones. La medición de la HA podría servir como indicador in vitro de la potencia del antiveneno, el antiveneno producido por el INS guarda las condiciones in vitro de inhibición de tres de las principales actividades de los venenos de serpientes peruanas.
The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some bi... more The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.
The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some bi... more The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.
Toxicon : official journal of the International Society on Toxinology, 2012
This communication describes the general biochemical properties and some immunological characteri... more This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD 50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.
Las serpientes Bothrops sp. causan el mayor número de casos de ofidismo en el Perú, su veneno con... more Las serpientes Bothrops sp. causan el mayor número de casos de ofidismo en el Perú, su veneno contiene enzimas que participan en la difusión de la ponzoña, así como en sus efectos miotóxicos, edemáticos y de alteración en la agregación plaquetaria. Objetivos. Evaluar el efecto del antiveneno botrópico polivalente al estado líquido producido por el Instituto Nacional de Salud (INS) sobre la fosfolipasa A 2 (PLA2), L-aminoácido oxidasa (LAO) y hialuronidasa (HA) de los venenos de B. atrox, B. barnetti, B. brazili y B. pictus. Materiales y métodos. La PLA2 fue determinada por el retardo en el tiempo de coagulación de una emulsión lipoproteica al 45%, LAO usando Lleucina como substrato en presencia de O-dianisidina y HA empleando ácido hialurónico y el reactivo turbidimétrico BCTA, se usó para cada enzima ½, 1 y 2 dosis del antiveneno al estado natural o calentado a 37 °C durante cinco días ensayados por triplicado. Resultados. HA fue la enzima más neutralizada por el antiveneno, todos los venenos con excepción de B. brazili fueron totalmente inhibidos a cualquier dosis. Para LAO se tuvieron valores de inhibición de 68 a 100% usando dos dosis del antiveneno, mientras que PLA2 fue la menos inhibida (70 a 80%) a dos dosis. Con el antiveneno calentado se registró una disminución del efecto inhibitorio encontrado inicialmente. Conclusiones. La medición de la HA podría servir como indicador in vitro de la potencia del antiveneno, el antiveneno producido por el INS guarda las condiciones in vitro de inhibición de tres de las principales actividades de los venenos de serpientes peruanas.
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