Mice plasma fibrinogen consumption by thrombin-like enzyme present in rattlesnake venom from the north-east region of Argentina

Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. [quot ]Gyroxin syndrome[quot ] in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.

Teniendo en cuenta la variabilidadde los componentes del veneno de serpientes de una misma especie que habitan regiones diferentes, se decidióestudiar las propiedades particulares del veneno de Crotalus durissus terrificus que habita el nordeste de Argentina, Giroxina, una enzima con actividad trombínica, fue aislada del veneno por cromatografía de filtración por gel y de afinidad; se comprobó su homogeneidad y se determinó su peso molecular, 33 kDa, por SDSPAGE. Se ensayó el síndrome giroxina en ratones, los que mostraron cambios en el comportamiento, confirmandoque la enzima tipo trombina aislada es giroxina. Se evaluó la acción de esta enzima sobre los niveles de fibrinógeno plasmático en ratones, comparándola con la del veneno crudo. Se comprobó que la enzima provoca una disminución de los niveles plasmáticos de fibrinógeno hasta la incoagulabilidad, durante la primer hora de inoculación, mientras que el veneno entero produjo una reducción del nivel plasmático en un 60%; sin embargo, en ambos casos, se evidenció una rápida reposición de fibrinógeno plasmático, alcanzando sus valores normales en un plazo de 10 horas. Se observó coagulación intravascular con la administración de giroxina una hora después de la inoculación, evidenciados en estudios histológicos de tejido pulmonar, cardíaco y hepático. En ensayos realizados in vitro, la enzima aislada mostró capacidad de degradar fibrinógeno como así también coágulos de fibrina, mientras que el veneno exhibió débil actividad hidrolítica sobre ambos sustratos. Es probable que esta baja actividad sea debida a la baja concentración de la enzima en el veneno. La disminución de los niveles de fibrinógeno plasmático observado en ratones se debería a la acción coagulante de la enzima, sin embargo no se descarta que también contribuya a este proceso una acción hidrolítica sobrefibrinógeno y fibrina por parte de la enzima.

Rocha e Silva, the snake venom and 50th anniversary of bradykinin discovery

In this article we emphasize the importante of the snake venom toxins for the development of the autopharmacology concept (Sir Henry Dale, 1933) and how they led to the discovery of bradykinin and to the development of the first active-site directed inhibitor of the angiotensin converting enzyme. We also describe the most recent development concerning the possible role of bradykinin potentiating peptides from Bothrops jararaca venom for the cardiovascular pharmacology. A fundamental step leading to what can be considered one of the most significant contributions given by Brazilian scientists to biomedical sciences, was originated in the efforts of Rocha e Silva to identify and characterize the actors involved in anaphylaxis. His main scientific motivation and efforts which influenced a whole generation of prominent Brazilian scientists, should be reminded when we are commemorating the 50th anniversary of the bradykinin discovery. One important consequence of the use of the Bothrops jararaca venom leading to the discovery of bradykinin was the development of captopril, the most successful drug used by milions of people all over the world to treat arterial hypertension. The purpose of this article is to dissect some of the historical background which aim to stress the need for the adequate scientific environment allowing the privileged scientific minds to uncover secrets of Nature for the benefit of mankind.

Hemorragia inducida por venenos de serpientes de Argentina / Hemorrhage induced by snake venoms in Argentina

Se inocularon ratones de 18 a 20 g de peso por vía intradérmica con 0.1 ml de diluciones seriadas de veneno en solución salina amortiguada a pH 7.2. Se conformaron grupos de 4 animales los que se sacrificaron a las 2 horas de la inoculación. Se quitó la piel y se midió el área hemorrágica. Se utilizaron los venenos de Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu y Bothrop neuwiedii. Todos ellos presentaron actividad hemorrágica. Crotalus durissus terrifcus no manifestó actividad hemorrágica.

Isolation and partial characterization of Lachesis muta melanocephala coagulant proteinase: biochemical parameters of the venom

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.

Generation of protective immune sera by Crotalus durissus terrificus venom detoxified by controlled iodination

1. Whole soluble venom from the snake Crotalus durissus terrificus was detoxified by controlled iodination. Doses equivalent to 100 LD50 of the native venom were administerd to mice, without signs of intoxication. 2. The non-toxic iodinated derivative were able to stimulate antibodies in rabbits and horses within a short period (6 months) of inmunization. Horse antisera attained titers of 0.5 to 0.9 mg/ml for protection aginst native venom. 3. Horse antisera obrained in horses from native and iodinated venom were run against both native and iodinated venoms, as antigens, in gel immunodiffusion. The precipitation lines showed total identity of the types of sera