SAXS study of crotapotin at low pH.

The structure of crotapotin, a protein extracted, from the venom of the Crotalus durissus terrificus, in solution at pH = 1.5, was studied by SAXS. The experimental results yield structural parameter values of the molecular radius of gyration Rg = 13.6 A, volume v = 16.2 x 10(3) A3 A3 and maximal dimension Dmax = 46 A. The distance distribution function deduced from the scattering measurements is consistent with an overall molecular shape of an oblate ellipsoid of revolution with asymmetry parameter v = 0.45.

Structure-function relationship for the highly toxic crotoxin from Crotalus durissus terrificus.

The three-dimensional structure of the highly toxic crotoxin from Crotalus durissus terrificus was modelled based on sequence analysis and the refined structure of calcium-free phospholipase of Crotalus atrox venom. Small-angle x-ray scattering experiments were performed on aqueous solutions of crotoxin. The radial distribution function derived from these scattering experiments and the one calculated from the model structure are in good agreement. Crotoxin consists of a basic and an acidic subunit. The model strongly suggests that the overall folding motif of phospholipases has been preserved in both subunits. The basic domain has an intact active site. The residues that are expected to contact the lipid tails of the phospholipid are different from other phospholipases, but they are all hydrophobic. The acidic domain consists of three independent chains interconnected by disulfide bonds. Compared to other phospholipases the active site for the greater part has been preserved in this domain, but it is not very well shielded from solvent. Most residues normally in contact with the lipid tails of the phospholipid are missing, which might explain the acidic subunit's lack of phospholipase activity. A homology between the third chain of the acidic domain and neurophysins suggests that the acidic domain may act as a chaperone for the basic domain.

SAXS study of the snake toxin alpha-crotamine.

alpha-crotamine is a small toxic protein (42 amino acid residues with three disulphide bridges) present in the venom of Crotallus durissus terrificus. Molecular parameters (Rg = 13.7 A, S = 3,000 A2, V = 9,200 A3 and Dmax = 40 A) were derived from SAXS curves obtained from a solution of this protein at pH = 4.5. An excellent agreement between the experimental distance distribution curve and that calculated from a model consisting of two lobes linked by the Cys(18)-Cys(30) disulphide bridge.

South American rattlesnake venom: its hemolytic power.

The hemolytic power of rattlesnake venom (Crotalus durissus terrificus) was studied. A high percentage of sample with negative hemolytic power was detected when sheep red blood cells were used. A large number of venoms with hemolytic power, though with a low hemolysis percentage, were detected when liquid, recently extracted venom was used. When crystallized venom was used under the same experimental conditions, a higher percentage of positivity for hemolysis was obtained. When the results obtained on agar plates were compared to those obtained in test tubes, a large number of animals with a higher percentage of hemolysis were detected, though this value was not proportional to the number of animals showing positive plate hemolysis. When the hemolytic power of these venoms was tested on human red blood cells, a large percentage of animals with venoms having a low hemolytic power was also detected. Hemolytic power was much greater when human red blood cells were tested with crystallized venom. The preparation of red blood cells also had an important effect and the use of red blood cells from defibrinated blood is recommended. We conclude that rattlesnake venom has hemolytic power that increases when the venom is crystallized. Red blood cells should be properly prepared for the lysis reactions. We suggest that the lytic power of the venom is related to venom concentration and to the purity of its fractions.

Raman and infrared studies on the conformation of porcine pancreatic and Crotalus durissus terrificus phospholipases A2.

Raman and infrared spectroscopies were used to investigate conformational features of Crotalus durissus terrificus and porcine pancreatic phospholipases A2, as well as the proenzyme of the latter. The results indicate that conformational changes occur for the phospholipase molecules as a consequence of different experimental conditions such as change of physical state, presence of certain ionic species and interaction with a model substrate analog. Amorphous and crystalline solid phospholipase present discrepant conformational features. Conformational transitions were detected for the pancreatic zymogen----phospholipase A2 transformation and different secondary contents were observed for a toxic and a nontoxic form of the phospholipase molecule. All those structural changes have been shown to involve primarily the architecture of the polypeptide backbone rather than the conformation of amino acid residue side-chains. Disulfide bridges have shown consistently a gauche-gauche-gauche geometry which has not been disturbed by any of the experimental conditions employed. The external occurrence of tryptophan residues has been a common feature for the systems assayed, as well as the predominant localization of tyrosine residues in hydrophilic environment, probably at the molecular surface.

Rattlesnake venom: action upon erythrocytes and leucocytes of rats.

We studied the action of whole rattlesnake venom on red blood cells and leucocytes of adult male and female rats. Animals were surgically cannulated for blood collection directly from the inferior caval vein and injected intramuscularly in the thigh with a mixture of venoms from a large number of rattlesnakes. The signs shown by the animals were paralysis of the hind part of the body, lack of motor coordination, and respiratory difficulties, with death occurring in some cases. Necroscopy showed petechial hemorrhage in the intestine and jejunum and darkening of the viscera, which was found to be due to engorged blood vessels upon histopathological examination. Blood examination showed a change in color to dark brown due to the transformation of hemoglobin to methemoglobin. Venom fractions were found to have a low hemolytic power because of their low concentration in the venom samples. Blood sedimentation rate showed a clear variation, especially 60 minutes after venom injection. Both phenomena may be linked to the lytic power of the venoms. An interesting phenomenon was that the animals showed initial leucopenia, which was followed by persistent leucocytosis. Lymphocytopenia and increased neutrophil numbers were also observed. The present results led us to conclude that rattlesnake venom has a relative hemolytic power which increases with venom concentration and with the concentration of the fractions in whole venom.

Rattlesnake venom: action upon erythrocytes and leucocytes of rats.

We studied the action of whole rattlesnake venom on red blood cells and leucocytes of adult male and female rats. Animals were surgically cannulated for blood collection directly from the inferior caval vein and injected intramuscularly in the thigh with a mixture of venoms from a large number of rattlesnakes. The signs shown by the animals were paralysis of the hind part of the body, lack of motor coordination, and respiratory difficulties, with death occurring in some cases. Necroscopy showed petechial hemorrhage in the intestine and jejunum and darkening of the viscera, which was found to be due to engorged blood vessels upon histopathological examination. Blood examination showed a change in color to dark brown due to the transformation of hemoglobin to methemoglobin. Venom fractions were found to have a low hemolytic power because of their low concentration in the venom samples. Blood sedimentation rate showed a clear variation, especially 60 minutes after venom injection. Both phenomena may be linked to the lytic power of the venoms. An interesting phenomenon was that the animals showed initial leucopenia, which was followed by persistent leucocytosis. Lymphocytopenia and increased neutrophil numbers were also observed. The present results led us to conclude that rattlesnake venom has a relative hemolytic power which increases with venom concentration and with the concentration of the fractions in whole venom.

Rattlesnake venom: action upon erythrocytes and leucocytes of rats

Hemos estudiado la acción del veneno completo de serpiente de cascabel sobre los glóbulos rojos y leucocitos de ratas adultas machos y hembras. Los animales fueron quirúrgicamente canulados para realizar recolecciones de sangre directamente de la vena cava inferior, e inyectados intramuscularmente en el muslo con la mezcla de veneno obtenido de un gran número de cascbeles. Los signos mostrados por los animales fueron parálisis de la parte posterior del cuerpo, déficit de la coordinación motora y dificultad repiratoria, con muerte en algunos casos. La autopsia mostró hemorragias petequiales en el intestino y yeyuno, además de oscurecimiento visceral y obstrucción de los vasos sanguíneos verificada en el examen histopatológico. Los exámenes sanguíneos mostrarón cambios de color hacia marrón oscuro debido a la transformación de la hemoglobina en metahemoglobina. Los índices de sedimentación sanguínea mostraron una clara variación, especialmente 60 minutos después de inuectado el veneno. Ambos fenómenos pueden estar ligados al poder lítico de los venenos. Un interesante fenómeno fue el que los animales presentaron inicialmente leucopenia, que fue seguida por persistente leucocitosis. Los presentes reultados nos llevan a concluir que el veneno de cascabel tiene un relativo poder hemolítico que se incrementa con la concentración del veneno y con la concentración de las fracciones en veneno completo (AU)

Rattlesnake venom: action upon erythrocytes and leucocytes of rats

Hemos estudiado la acción del veneno completo de serpiente de cascabel sobre los glóbulos rojos y leucocitos de ratas adultas machos y hembras. Los animales fueron quirúrgicamente canulados para realizar recolecciones de sangre directamente de la vena cava inferior, e inyectados intramuscularmente en el muslo con la mezcla de veneno obtenido de un gran número de cascbeles. Los signos mostrados por los animales fueron parálisis de la parte posterior del cuerpo, déficit de la coordinación motora y dificultad repiratoria, con muerte en algunos casos. La autopsia mostró hemorragias petequiales en el intestino y yeyuno, además de oscurecimiento visceral y obstrucción de los vasos sanguíneos verificada en el examen histopatológico. Los exámenes sanguíneos mostrarón cambios de color hacia marrón oscuro debido a la transformación de la hemoglobina en metahemoglobina. Los índices de sedimentación sanguínea mostraron una clara variación, especialmente 60 minutos después de inuectado el veneno. Ambos fenómenos pueden estar ligados al poder lítico de los venenos. Un interesante fenómeno fue el que los animales presentaron inicialmente leucopenia, que fue seguida por persistente leucocitosis. Los presentes reultados nos llevan a concluir que el veneno de cascabel tiene un relativo poder hemolítico que se incrementa con la concentración del veneno y con la concentración de las fracciones en veneno completo

A study of bacterial contamination of rattlesnake venom

Os autores estudaram a contaminaçäo bacteriana do veneno de cascavéis mantidos em cativeiro e das recentemente capturadas. Verificaram que os venenos dos animais cativos apresentaram alta incidência de contaminaçäo e os tipos como recentemente capturados estavam com baixa contaminaçäo aparente

Dialyzable transfer factor in experimental Chagas' disease: in vitro studies.

The dialyzable transfer factor (TF) was prepared from spleen and lymph node cells of either normal (TFn) or infected (TFi) mice with Trypanosoma cruzi. The ability of TFn and TFi in transferring cellular immunity to T. cruzi antigens was assessed by the macrophage migration inhibition assay and lymphocyte transformation test. The results obtained with these two immunological assays indicated that only TFi is able to transfer cellular immune responses. This phenomenon was antigen specific. The content of free amino acids in TFi preparation was higher than in TFn. However, our data indicated that the stimulation of lymphocyte transformation is not due to the increase in glycine and serine. This activity of TFi required the addition of T. cruzi antigens. Our findings support the hypothesis that TFi is derived from immune RNA.

Saxs study of structure and conformational changes of crotamine.

The radius of gyration of crotamine is determined by the small angle x-ray scattering technique. Several molecular solutions have been studied to correct for concentration effects. The apparent molecular radius of gyration is also determined as a function of pH. An important change between pH 9.5 and 12.5 is attributed to a dominant effect of molecular aggregation.

Myonecrosis, myoglobinuria and acute renal failure induced by South American rattlesnake (Crotalus durissus terrificus) envenomation in Brazil.

The venom of the Brazilian rattlesnake Crotalus durissus terrificus is know to have hemolytic and neurotoxic physiopathological activities which may cause acute renal failure with hemoglobinuria and/or methemoglobinuria. As far as we know, no report has been published on the ability of the venom of this rattlesnake species to cause rhabdomyolysis. In the present paper we demonstrate that the venom of Brazilian snakes of the genus Crotalus can induce systemic myonecrosis. Clinical, laboratory and anatomo-pathological data for two patients referred to the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, 24 hr after a rattlesnake bite, are presented. In both cases, exaggerated elevation of serum levels of the enzymes creatine phosphokinase, lactate dehydrogenase and glutamic-oxaloacetic transaminase were detected, as well as data suggesting acute hypercatabolic renal failure. Immunoelectrophoresis of the serum and urine of these patients, carried out against specific anti-myoglobin serum (Behringwerke), demonstrated myoglobinemia and myoglobinuria, confirming injury to muscle tissue. Electron microscopy of a calf muscle biopsy taken from the leg contralateral to the bite from one patient revealed foci of myonecrosis.

Isolation and characterization of toxic proteins from the venom of the Brazilian scorpion Tityus serrulatus.

Through gel filtration on Sephadex G-25 and chromatography on CM-cellulose-52 five toxic proteins, electrophoretically pure, were isolated from the venom of the Brazilian scorpion Tityus serrulatus and partially characterized, as follows: 1. Toxin T1 VIII, with 61 amino acid residues, mol. wt 6675 and amino terminal sequence Lys-Glx-Gly-Tyr-Leu-Met-Asx-His-Glx-Gly-Cys-Lys-; 2. Toxin T1VI with amino acid residues, mol. wt. 7549 and amino terminal sequence Gly-His-Phe-Gly-Lys; 3. Toxin T2III(I), with 63 amino acid residues, mol. wt. 7216 and amino terminal sequence Lys-Lys-Asx-Gly-Tyr-Pro-Val-Cys-Cys-Ser-; 4. Toxin T2IV, which is apparently identical to toxin T1VIII above, since it showed the same elution volume in chromatography on CM-cellulose-52, the same N-terminal Lys and the same electrophoretic mobility as T1VIII; 5. Toxin T1IV, a not previously described toxin from the venom of T. serrulatus, with 45 amino acid residues, mol. wt. 5188 and amino terminal sequence Lys-Glx-Gly-Tyr-Leu-, identical to the first five residues of T1VIII, although with a lower molecular weight. The pharmacological study of T1VIII in guinea pig vas deferens showed a pre-junctional sensitizing action, evidenced by a decrease of the dose-response curves to adrenaline and acetylcholine, with no increase of the maximum. This effect may be due to the liberation of noradrenaline.

[The primary structure of crotamine (author's transl)]. / Die Primärstruktur des Crotamins

The primary structure of crotamine, a basic toxin isolated from the venom of the South American rattle-snake Crotalus durissus terrificus has been determined. The polypeptide chain is composed of 42 residues of amino acids. Crotamine shows a molecular weight of 4900 and contains 6 half cystine, 9 lysine, 2 arginine, 2 histidine and 2 tryptophan residues.