RESUMO
The present study aimed to evaluate the effects of two serine proteases from Bothrops pirajai snake venom, named BpirSP27 and BpirSP41, on the complement system and the inflammatory response. The effects of these enzymes on the human complement system were assessed by kinetic hemolytic assays, evaluating the hemolysis promoted by the classical/lectin (CP/LP) and alternative (AP) pathways after incubation of normal human serum with the serine proteases. The results suggested that these enzymes were able to induce modulation of CP/LP and AP at different levels: BpirSP41 showed higher inhibitory effects on the hemolytic activity of CP/LP than BpirSP27, with inhibition values close to 40% and 20%, respectively, for the highest concentration assayed. Regarding AP, both enzymes showed percentages of inhibition of the hemolytic activity around 20% for the highest concentrations tested, indicating similar effects on this complement pathway. The proinflammatory effects of B. pirajai serine proteases were evaluated regarding their ability to induce paw edema, variations in the pain threshold and leukocyte recruitment at the site of injection. Both showed mild effects on these inflammatory processes, leading to low levels of increase of paw volumes and decrease in pain thresholds in rats up to 6 h after injection, and inducing neutrophil recruitment without significant increases in the total number of leukocytes in the inflammatory exudates after 6 and 24 h of administration into mice peritoneal cavity. These results suggest that serine proteases must present a minor role in the inflammation caused by B. pirajai snake venom.
Assuntos
Bothrops , Ativação do Complemento/efeitos dos fármacos , Venenos de Crotalídeos/enzimologia , Edema/induzido quimicamente , Hiperalgesia/induzido quimicamente , Serina Proteases/toxicidade , Adulto , Animais , Células Cultivadas , Ativação do Complemento/imunologia , Venenos de Crotalídeos/química , Edema/sangue , Edema/imunologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Exsudatos e Transudatos/citologia , Exsudatos e Transudatos/imunologia , Feminino , Hemólise/efeitos dos fármacos , Humanos , Hiperalgesia/sangue , Hiperalgesia/imunologia , Contagem de Leucócitos , Leucócitos/citologia , Leucócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Cavidade Peritoneal/citologia , Coelhos , Ratos , Ratos Wistar , Serina Proteases/isolamento & purificação , Soro/imunologia , Ovinos , Adulto JovemRESUMO
A fibrino(geno)lytic nonhemorrhagic metalloproteinase (BleucMP) was purified from Bothrops leucurus snake venom by two chromatographic steps procedure on DEAE-Sephadex A-25 followed by CM-Sepharose Fast Flow column. BleucMP represented 1.75% (w/w) of the crude venom and was homogeneous on SDS-PAGE. BleucMP analyzed by MALDI TOF/TOF, showed a molecular mass of 23,057.54Da and when alkylated and reduced, the mass is 23,830.40Da. Their peptides analyzed in MS (MALDI TOF\TOF) showed significant score when compared with those of other proteins by NCBI-BLAST2 alignment display. As regards their proteolytic activities, BleucMP efficiently acted on fibrinogen, fibrin, and was inhibited by EDTA and 1.10-phenanthroline. This enzyme was also able to decrease significantly the plasma fibrinogen level provoking blood incoagulability, however was devoid of hemorrhagic activity when tested in the mice skin and did not induce relevant biochemical, hematological and histopathological alterations in mice. The aspects addressed in this paper provide data on the effect of BleucMP in envenomation from B. leucurus snakes in order to better understand the effects caused by snake venom metalloproteinase.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Metaloendopeptidases/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia/métodos , Venenos de Crotalídeos/química , Fibrinólise/efeitos dos fármacos , Hemorragia/induzido quimicamente , Hemorragia/patologia , Humanos , Masculino , Metaloendopeptidases/toxicidade , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Mapeamento de Peptídeos , Pele/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Testes de ToxicidadeRESUMO
Phospholipase A2 (PLA2, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A2 from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE-Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl-Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA2s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA2s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.