RESUMO
The fibrinogenolytic activity of Lachesis muta stenophyrs venom was studied. Wistar rats catheterized at carotid artery and jugular vein were inoculated with crude venom or enzyme and changes in arterial pressure, cardiac frequency and electrocardiogram were monitored. The enzyme induced a greater fibrinogen reduction than the crude venom without any cardiovascular or histological alteration. In vitro crude venom coagulated blood whereas the enzyme reduced fibrinogen in 23%. Results suggest the potential use of the fibrinogenolytic enzyme as antithrombotic agent.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fibrinogênio/farmacologia , Fibrinólise , Venenos de Víboras/toxicidade , Viperidae , Análise de Variância , Animais , Pressão Sanguínea/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Dose Letal Mediana , Masculino , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Ratos , Ratos Wistar , Fatores de Tempo , Venenos de Víboras/enzimologiaRESUMO
The primary structure of the lectin-like protein from Lachesis muta stenophyrs venom was deduced from analysis of the N-terminus and the sequence of peptides obtained after digestion with trypsin, Arg-C enzyme, Staphylococcus aureus V8 protease and endoproteinase Asp-N. Peptides generated by cleavage of the lectin with cyanogen bromide and o-iodosobenzoic acid were also sequenced. Comparison of the complete 135 amino acid residues sequence with those of the lectin from the venom of Crotalus atrox, with platelet coagglutinin from Bothrops jararaca beta-fragment and with the anticoagulant B protein chain from Trimeresurus flavoviridis venom, revealed 92, 46 and 29% identity, respectively. Significant homology was also found with C-type carbohydrate-recognition domain-like structures from invertebrate and vertebrate lectins. To our knowledge, this is the second known primary structure of a lectin-like protein from snake venom.
Assuntos
Venenos de Crotalídeos/química , Sequência de Aminoácidos , Animais , Bothrops , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/genética , Lectinas/química , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Staphylococcus aureus/enzimologia , Tripsina/metabolismo , ViperidaeRESUMO
A proteolytic enzyme from L. muta stenophrys was isolated by gel filtration on Bio Gel P-100 followed by FPLC on MONO S column. The enzyme exhibited proteolytic activity toward casein, hemoglobin and fibrinogen with a pH optimum around 10. The activity was inhibited by EDTA while trypsin inhibitors were not inhibitory. It is a glycoprotein, Mr 14 kDa with a high content of Asp, Glu, and Leu residues and a low content of Cys and Trp. The protease is devoid of myotoxic, hemorrhagic, esterolytic and amidolytic activities. It lyses the alfa and beta chains of human fibrinogen and releases kinin from L.M.W. kininogen. No release of histamine was observed upon incubation with mast cells.
Assuntos
Venenos de Crotalídeos/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Animais , Cromatografia em Gel , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologiaRESUMO
A lectin was isolated from the crude extract prepared from the seeds of E. costaricensis. It was purified by gel filtration on Sephadex G-100 followed by DEAE-Sephadex A-50 chromatography. PAGE revealed only one protein band, while analytical isoelectric focusing revealed four bands. The protein is a dimer with M(r) 58kda not united by disulfide bridges. It is a glycoprotein with 6.5% of neutral sugars, stable at 70 degrees C and at a pH range between 2 to 10. The protein exhibited a non specific agglutination of human erythrocytes, nevertheless it differentiated between erythrocytes of animal origin, agglutinating those of rabbit and chicken and not those from horse, goat, sheep or rat. Galactose, N-acetyl-D-galactosamine, lactose and EDTA are inhibitors while Ca++ and Mn++ acted as activators of the agglutination. No change in the blood pressure was observed when the lectin was intravenously injected in rats.
Assuntos
Erythrina/química , Lectinas/isolamento & purificação , Plantas Medicinais , Animais , Antígenos de Grupos Sanguíneos , Galinhas , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Hemaglutinação , Humanos , Focalização Isoelétrica , Lectinas de Plantas , Coelhos , RatosRESUMO
La lectina de la semilla de E. costaricensis se purificó a partir del extracto crudo mediante filtración por Sephadex G-100 y cromatografía por DEAE-Sephadex A-50. La electroforesis en gel de acrilamida (PAGE) demostró la presencia de una única banda proteica. El isoelectroenfoque analítico demostró la presencia de cuatro isolectinas. La masa relativa obtenida por filtración en Sephadex fue de 58 KDa y por PAGE en condiciones reductoras de 29.5 KDa. La proteína es fundamentalmente un dímero no unido por enlaces disulfuro, con un contenido de 6.5% de azúcares neutros. Es estable hasta 70-C y en un àmbito de pH de 2 a 10. Aglutina indistintamente los eritrocitos humanos y diferencia entre eritrocitos de origen animal, aglutinando los de conejo y pollo y no los de caballo, cabra, carnero ni rata. La galactosa N-acetil-galactosamina, lactosa y el EDTA inhiben su acción aglutinante, los iones calcio y mangneso son activadores. No se encontró efecto vasopresor al inyectar la lectina itravenosamente en ratas
Assuntos
Humanos , Animais , Coelhos , Ratos , Erythrina/química , Lectinas/isolamento & purificação , Galinhas , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Antígenos de Grupos Sanguíneos , Hemaglutinação , Focalização Isoelétrica , Extratos VegetaisRESUMO
A lectin-like protein was isolated from L. muta venom by gel filtration on BIO Gel P-100 followed by column Chromatography on DEAE-sephades A-50. The protein eluted at 0.4 M Nacl in 0.01 Tris pH 7.3 and exhibited agglutinin activity toward 0+ human erythrocytes. The protein is a dimer with Mr 28 kDa. Amino acid analysis revealed high content of tryptophan and acid recidues and low content of cysteine and methionine residues. No neutral carbohydrates and sialic acid were detected. Circular dichroic spectrum shows 78% of B structure and 1% of alpha structure. In vitro experiments with erythrocytes from rat, rabbit and dog revealed strong agglutination while red blood cells from mice, sheep and goat were not agglutinated. In vivo experiments using anesthetized rats, a sharp and prolonged fall in the blood pressure was observed at protein dose of 1.5 mg/kg. Double dose of protein caused the death of the animal.
Assuntos
Venenos de Crotalídeos/química , Hemaglutininas/isolamento & purificação , Lectinas/isolamento & purificação , Viperidae/metabolismo , Aminoácidos/análise , Animais , Cães , Eritrócitos/efeitos dos fármacos , Cabras/sangue , Hemaglutininas/química , Hemaglutininas/farmacologia , Hemaglutininas/toxicidade , Humanos , Hipotensão/induzido quimicamente , Lectinas/química , Lectinas/farmacologia , Lectinas/toxicidade , Camundongos , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/isolamento & purificação , Proteínas/farmacologia , Proteínas/toxicidade , Coelhos , Ratos , Ratos Sprague-Dawley , Ovinos/sangue , Especificidade da EspécieRESUMO
A lectin-like protein was isolated from L. muta venom by gel filtration on BIO Gel P-100 followed by column Chromatography on DEAE-Sephadex A-50. The protein eluted at 0.4 M NaCl in 0.01 Tris pH 7.3 and exhibited agglutinin activity toward 0**+ human erytrocytes. The protein is a dimer with Mr 28 kDa. Amino acid analysis revealed high content or tryptophan and acid recidues and low content of cysteine and methionine residues. No neural carbohydrates an sialic acid were detected. Circular dichroic spectrum shows 78% of B structure and 1% of alfa structure. In vitro experiments with ery throcytes from rat, rabbit and dog revealed strong agglutination while red blood cells from mice, sheep and goat were not agglutinated. In vivo experiments using non-anesthetized rats, a sharp and prolonged fall in the blood pressure was observed at protein dose of 1.5 mg/kg. Double dose of protein caused the death of the animal
Assuntos
Humanos , Cães , Camundongos , Coelhos , Ratos , Animais , Aminoácidos/análise , Técnicas In Vitro , Proteínas/isolamento & purificação , Venenos de Serpentes/análise , Aglutinação , Costa Rica , Eletroforese em Gel de Poliacrilamida , Cabras , Ratos Endogâmicos , Ovinos , Cloreto de Sódio/análiseRESUMO
The coagulant proteinase of L. m. melanocephala was purified by DEAE Sephadex A-50 followed by agmatine CH- Sepharose and gel filtration on Sephadex G-100. The enzyme exhibited many of the properties ascribed to "mudasa", the coagulant proteinase from L. m. stenophirs venom. Its molecular weight by gel filtration was 35 kDa and its specific clotting activity was 702 NIH/mg of protein. A 32 fold increase in the clotting activity was obtained by purification. The coagulant proteinase exhibited esterolytic activities toward lysine and arginine esters as well as amidolytic activity. Significant differences are observed when compared with the activities of "mudasa", the former is less esterolytic and amidolytic, although its activity toward TLEME is higher. Significant differences in the activities are also observed when the venom from the Pacific and Atlantic L. muta populations (corresponding to the subspecies L. m. melanocefala and L. m. stenophyrs) are compared toward the same substrates. The Pacific type is less amidolytic and more esterolytic toward BAME and BAEE, although toward the lysine and tyrosine esters no significant differences are observed. The venom from the Pacific population is more coagulant and less proteolytic than the venom from the Atlantic population. Analytical isoelectric focusing of both populations of venom revealed important differences in the number and intensity of the protein bands. The results here given further substantiate the taxonomical differentiation already given to the Pacific and Atlantic Costa Rican population of L. muta.
Assuntos
Venenos de Crotalídeos/metabolismo , Endopeptidases/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Fracionamento Químico , Cromatografia em Gel , Costa Rica , Endopeptidases/farmacologia , Focalização IsoelétricaRESUMO
The coagulant proteinase of L. m. melanocephala was purified by DEAE Sephadex A-50 followed by agmatine CH- Sepharose and gel filtration on Sephadex G-100. The enzyme exhibited m,any of the properties adscribed to -mudasa-, the coagulant proteinase from L. m. stenophirs venom. Its molecular weight by gel filtration was 35 kDa and its specific clotting activity was 702 NIH/mg of protein. A 32 fold increase in the clotting was obtained by purification. The coagulant proteinase exibited esterolytic activities toward lysine and arginine esters as well as amidolytic. Significant differences are observed when compared with the activities of -mudasa-, the former is less esterolytic, although its activity toward TLEME is higher. Significant differences in the activities are also observed when the venom from the Pacific and Atlantic L. muta populations (corresponding to the subspecies L.m. melanocefala and L.m. Stenophyrs) are compared toward the same substrates. The Pacific type is less amidolytic and more esterolytic toward BAME and BAEE, although toward the lysine and tyrosine esters no significant differences are observed. The venon from the Pacific populations is more coagulant and less proteolytic than the venom from the Atlantic population. Analytical isoelectric focusing of both populations of venom revealed important differences in the number and intensity of the protein bands. The results here given further substantiate the taxonomical differentiation already given to the Pacific and Atlantic Costa Rican population of L. muta.
Assuntos
Criança , Feminino , Masculino , Pessoa de Meia-Idade , Venenos de Crotalídeos/metabolismo , Endopeptidases/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Fracionamento Químico , Cromatografia em Gel , Costa Rica , Endopeptidases/farmacologia , Focalização IsoelétricaRESUMO
Lachesis muta snake venom induced aggregation of bromelain sensitized human erythrocytes at a concentration of 1 mg/ml. The hemagglutinating protein was purified by DEAE-Sephadex A-50 column chromatography. Polyacrylamide gel electrophoresis revealed at least three bands, whereas SDS electrophoresis in the presence of 2-mercaptoethanol showed a single one. Isoelectric focusing revealed hemagglutinating activity in the range of pH 3-8. The maximum peak (mutina) at pH 5.5. This fraction was active in agglutinating human RBC of types A, B, O Rh (+) and B, O Rh (-). One mM EDTA and 1 mM Ca++ did not alter the agglutinating time significantly. Lactose and inositol inhibited the agglutination of A, B, O Rh (+) and B, O Rh (-) human RBC. The present study showed the non specificity of the hemagglutinating activity of mutina. It was also shown that mutina is a non-mitogenic protein.
Assuntos
Venenos de Crotalídeos/análise , Lectinas/isolamento & purificação , Aglutinação , Animais , Fracionamento Químico , Eritrócitos/imunologia , Testes de Hemaglutinação , Agregação PlaquetáriaRESUMO
The venom of Lachesis muta is a rich source of a thrombin-like enzyme. Its coagulant proteinase was purified by DEAE -Sephadex A -50 followed by agmatine CH -Sepharose and gel filtration on Sephadex G-100. On polyacrylamide gel electrophoresis at pH 8.4 a single band was observed. Its molecular weight by gel filtration was 49,000. The coagulant and esterolytic activities toward human fibrinogen and Tame of the inudasa were 662 NIH units/mg of protein and 4.37 delta OD225/min x 10(-3)/micrograms/ml, respectively. These values represent 23 and 5.7 fold increase over the crude venom. The enzyme mudasa, was evaluated with serum from human patients at Hospital Nacional de Niños Dr. Carlos Sáenz Herrera and found to be a valuable reagent for the quantification of fibrinogen on heparinized plasma.