Effect of exposition to chlorpyrifos upon plasmatic cholinesterases, hematology and blood biochemistry values in Bothrops asper (Serpentes: Viperidae).

The terciopelo (Bothrops asper), is one of the most important venomous snakes in Costa Rica and common on agriculture where insecticides are frequently used for pest control. To assess the exposure to organophosphates on captive B. asper, an experiment using chlorpyrifos and butyrylcholinesterase (BChE), as a biomarker was conducted. In addition to BChE, hematology, aspartate aminotransferase (AST), total proteins (TP) and albumin were measured after exposure. Different concentrations of chlorpyrifos were used in Group A (0.1%) and B (1%), while the Control Group received distilled water; each group was composed of 5 snakes. Values of BChE, AST, TP, and albumin were measured before exposure, and at 6, 12, 24, 196, 360 and 528 h post-exposure. Hematology values were measured after 24 h post-exposure. As result, an important variation between subjects in all groups before exposure was obtained. Moreover, BChE activity showed 37% inhibition of Group A when compared to Control Group at 12 h post-exposure, and a higher inhibition of Group B (97%) related to Control Group, at 6 h post-exposure. Recovery of BChE occurred towards 528 h, never reaching initial values. Despite some variation in the rest of parameters used, a marked relative lymphopenia and monocytosis occurred at 24 h, assuming stress as the main cause.

Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum.

Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation.

Aspectos citoquímicos e morfológicos de elementos sanguíneos das serpentes dos gêneros Bothrops e Crotalus mantidas em cativeiro no serpentário do Instituto Vital Brasil / Cytochemical and morphological aspects of blood cells from Bothrops and Crotalus kept in captivity in the serpentarium of the Institute Vital Brazil

A criação de serpentes peçonhentas em cativeiro vem se tornando prática cada vez mais difundida no país. Dessa forma, o conhecimento do manejo e da clínica de serpentes se torna prioritário, a fim de permitir maior sobrevida dos animais. No que concerne a serpentes peçonhentas, dados hematológicos já foram descritos na literatura, no entanto, apesar dos recursos utilizados, os dados analisados ainda são insipientes. Com o objetivo de caracterizar, morfologicamente, as células sanguíneas e de esclarecer diferenças morfológicas e funcionais, foram coletadas amostras de sangue de 50 serpentes pertencentes ao plantel do Instituto Vital Brasil. Foram confeccionados e analisados por microscopia óptica e citoquimicamente os esfregaços sanguíneos corados por métodos de Romanowsky e citoquímicos. Foi possível diferenciar as células, caracterizar e confirmar a existência de eosinófilo em B. atrox e C. durissus. Concluiu-se que a caracterização celular pode fornecer evidências indispensáveis ao entendimento da fisiologia de serpentes.(AU)

Breeding of venomous snakes in captivity is becoming increasingly widespread in the country, so clinical and management knowledge on these animals has become priority to increase survival of animals. Regarding venomous snakes, hematological data have been described in some studies; however, despite the resources used, data analyzed are still unrecognized. Aiming to characterize morphology of blood cells and clarify morphological and functional differences, blood samples were collected from 50 snakes belonging to the Instituto Vital Brazil squad. Blood smears were prepared and analyzed by optical microscopy and cytochemistrically, stained by Romanowsky and cytochemical methods. Cell differentiation was possible as well as characterization and confirmation of eosinophil in B. atrox and C. durissus. In conclusion, cell characterization can provide vital evidence to the understanding of the physiology of snakes.(AU)

Isolation and characterization of a novel metalloprotease inhibitor from Bothrops alternatus snake serum.

Resistance of snakes and some other animals to snake envenomation has been attributed to soluble factors present in their tissues. Here we report the isolation of a novel metalloprotease inhibitor from Bothrops alternatus snake serum (named BaltMPI) with high purity, using a four-step chromatographic method. BaltMPI has molecular weights of 60.5 and 42.4kDa, as determined by SDS-PAGE and mass spectrometry, respectively, and pI=5.27. The first 60 amino acids from the N-terminal region of BaltMPI, determined by Edman's degradation, showed high homology (97%) with the snake venom metalloprotease inhibitor (SVMPI) BJ46a and other SVMPIs (78-82%). The chromatographic fractions and purified BaltMPI exhibited anti-hemorrhagic activity against Batroxase and BjussuMP-I. BaltMPI was stable over wide ranges of pH (1, 5, 8, and 9) and temperature (-80, -20, 4, 60, and 100°C), and suppressed the fibrinogenolytic, fibrinolytic, and azocaseinolytic activities of Batroxase. BaltMPI specifically inhibited the activity of metalloproteases, without affecting the activity of serine proteases. Together, our results suggest that BaltMPI and other SVMPIs are promising molecules for the treatment of snake envenomation, in particular that caused by Bothrops sp.

Flow cytometric characterization of peripheral blood leukocyte populations of 3 neotropical snake species: Boa constrictor, Bothrops jararaca, and Crotalus durissus.

BACKGROUND: The reptilian immune system is represented by innate, humoral, and cell-mediated mechanisms, involving different types of blood leukocytes. The development of optimized methods for the advanced study of origin and function of reptilian blood leukocytes is needed. OBJECTIVES: The purpose of the study was to optimize leukocyte density gradient isolation protocols from snake peripheral blood samples, and characterize recovered cells by flow cytometry based on size and internal complexity for a qualitative and semi-quantitative assessment of leukocyte populations in one boa (Boa constrictor), and 2 viper species (Bothrops jararaca, Crotalus durissus). METHODS: Blood samples from 30 snakes (10 from each species, 5 males and 5 females) were collected in tubes with sodium heparin. Fresh blood was centrifuged with either ficoll-paque PLUS or percoll density gradients for leukocyte isolation. Flow cytometric leukocyte gates were defined based on size (forward scatter [FSC]) and internal complexity (side scatter [SSC]). Relative leukocyte differential counts after sorting the cells in these gates in one snake for each species were compared to conventional light microscopic differential counts on unsorted isolated leukocytes. RESULTS: There was no statistical difference in the relative leukocyte populations, including heterophils, azurophils, and small and large lymphocytes between samples isolated by ficoll or percoll. Four leukocyte gates were identified based on their location in FSC/SSC cytograms. The relative leukocyte differential counts after sorting in single animals showed some agreement with the light microscopy differential count on unsorted cells. CONCLUSIONS: Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays.

Depletion of plasma albumin for proteomic analysis of Bothrops jararaca snake plasma.

The proteomic analysis of plasma samples represents a challenge as a result of the presence of highly abundant proteins such as albumin. To enable the detection of biomarkers, which are commonly low-abundance proteins, in complex blood fluids, it is necessary to remove high-abundance proteins efficiently. Moreover, there is a range of about 10 orders of magnitude for the abundance of different protein species in serum. Here, we describe for the first time a study of reptilian albumin depletion using resins usually used in mammalian plasma depletion procedures. We performed the depletion of albumin from Bothrops jaraca plasma using the HiTrap Blue high-performance column (GE Healthcare Life Sciences, Piscataway, NJ, USA) and the kit Albumin & IgG Depletion SpinTrap column (GE Healthcare Life Sciences). In addition, proteomic approaches were used to analyze reptilian plasma. Our results showed that B. jararaca albumin bound to both columns, but those interactions were not enough to remove a large amount of albumin to reach an enrichment of low-abundance proteins. Although the depletion techniques used in this work were not the best to remove B. jararaca plasma albumin, our present work highlights the similarity between B. jararaca and mammalian albumin, contributing to the knowledge of comparative hemostatic proteins.

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma.

Phospholipases A(2) (PLA(2)s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA(2) inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA(2)s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated αBaltMIP, showed it to be a glycoprotein with Mr of ~24,000 for the monomeric subunit. CD spectra of the PLA(2)/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of αBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization.

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: structural and functional characterization.

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alphaBjussuMIP, showed it to be an oligomeric glycoprotein with M(r) of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alphaBjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alphaBjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alphaPLIs. alphaBjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alphaBjussuMIP may prove useful in the treatment of snakebite envenomations.

Identification of the B subtype of gamma-phospholipase A2 inhibitor from Protobothrops flavoviridis serum and molecular evolution of snake serum phospholipase A2 inhibitors.

A cDNA encoding a novel phospholipase A(2) (PLA(2)) inhibitor (PLI) was isolated from a Protobothrops flavoviridis snake (Tokunoshima island, Japan) liver cDNA library. This cDNA encoded a signal peptide of 19 amino acids followed by a mature protein of 181 amino acids. Its N-terminal amino acid sequence was completely in accord with that of a PLI, named PLI-II, previously found in P. flavoviridis serum. PLI-II showed a high similarity in sequence to the B subtype of gammaPLI, denoted gammaPLI-B, isolated from Agkistrodon blomhoffii siniticus serum. Thus, PLI-II is P. flavoviridis serum gammaPLI-B. Since PLI-I, previously isolated from P. flavoviridis serum, can be assigned as gammaPLI-A, P. flavoviridis serum contains both A and B subtypes of gammaPLI. Phylogenetic analysis of gammaPLIs from the sera of various kinds of snakes, Elapinae, Colubrinae, Laticaudinae, Acanthophiinae, Crotalinae, and Pythonidae, based on the amino acid sequences revealed that A and B subtypes of gammaPLIs are clearly separated from each other. It was also found that phylogenetic topologies of gammaPLIs are in good agreement with speciation processes of snakes. The BLAST search followed by analyses with particular Internet search engines of proteins with Cys/loop frameworks similar to those of PLI-II and PLI-I revealed that gammaPLI-Bs, including PLI-II and PLI-II-like proteins from mammalian sources, form a novel PLI-II family which possesses the common Cys/loop frameworks in the anterior and posterior three-finger motifs in the molecules. Several lines of evidence suggest that PLI-II is evolutionarily ancestral to PLI-I.

Identification of proteins similar to Bothrops jararaca coagulation inhibitor (BjI) in the plasmas of Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus snakes.

Bothrops jararaca coagulation inhibitor (BjI), a protein isolated from B. jararaca plasma, specifically inhibits the coagulant activity of thrombin. Our group previously identified proteins similar to BjI in the plasma of other snakes [Tanaka-Azevedo, A.M., Tanaka, A.S., Sano-Martins I.S., 2003. A new blood coagulation inhibitor from the snake Bothrops jararaca plasma: isolation and characterization. Biochem Biophys Res Commun 308, 706-712.]. In the present study, we analyzed the presence of BjI-like proteins in the plasmas of three different species of viperid snakes, Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus. These proteins exhibited 109 and/or 138 kDa and were immunologically related to BjI. They also inhibited the coagulant activity of thrombin, evaluated by the thrombin time test. These findings demonstrate the presence of proteins similar to BjI in these three species, although such inhibitor could not be observed in all samples of the specimens tested. Moreover, the presence of these proteins in the plasma is related to prolongation of thrombin time, implying a relationship between these proteins and their inhibitory coagulant activity upon thrombin. Our results suggest that BjI-like proteins are widely distributed among Crotalinae snakes found in Brazil.

Hypothalamic activity during altered salt and water balance in the snake Bothrops jararaca.

The effects of water and salt overload on the activities of the supraoptic and paraventricular nuclei and the adjacent periventricular zone of the hypothalamus of the snake Bothrops jararaca were investigated by measurements of Fos-like immunoreactivity (Fos-ir). Both water and salt overload resulted in changes in body mass, plasma osmolality, and plasma concentrations of sodium, potassium, and chloride. Hyper-osmolality increased Fos immunoreactivity in the rostral supraoptic nucleus (SON), the paraventricular nucleus (PVN), and adjacent periventricular areas. Both hyper- and hypo-osmolality increased Fos immunoreactivity in the intermediate SON, but not in other areas of the hypothalamus. Immunostaining was abundant in cerebrospinal fluid (CSF)-contacting tanycyte-like cells in the ependymal layer of the third ventricle. These data highlight some features of regional distribution of Fos immunoreactivity that are consistent with vasotocin functioning as a hormone, and support the role of hypothalamic structures in the response to disruption of salt and water balance in this snake.

BJ46a, a snake venom metalloproteinase inhibitor. Isolation, characterization, cloning and insights into its mechanism of action.

Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4-reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition.

Hematological values of some Bothrops species (Ophidia - Crotalidae) in captivity

Blood samples of 50 healthy specimens from each of the following species: Bothrops alternatus, Bothrops jararacussu, Bothrops moojeni, and Bothrops neuwiedi diporus all kept in captivity were taken to determine the hematocrit (PCV) value, red blood cell count (RBC), total leukocyte (WBC) and differential leukocyte count, thrombocyte count, mean corpuscular volume (MCV), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). These hematological parameters were compared to those obtained from other Bothrops species. PCV values, RBC, hemoglobin, WBC count, and differential leukocyte count are within the range of values reported for other Bothrops species, while the thrombocyte count was significantly lower. All the hematological parameters obtained from the four studied Bothrops species were higher than those described for B. ammodytoides.

Plasma arginine-vasotocin and hydroosmotic status of the terrestrial pit viper Bothrops jararaca.

Peaks corresponding to arg-vasotocin obtained by HPLC from Sep-Pak C18 column extracts of Bothrops jararaca plasma were identified by radioimmunoassay and amino acid analysis. Plasma vasotocin and protein levels, osmolality, and L-cystine-di-beta-naphthylamidase were also compared in snakes under normal hydration conditions with or without chronic administration of vasotocin or in the presence of chronic hydroosmotic challenges. Sep-Pak C18 and radioimmunoassay were validated for the extraction and determination of this peptide, respectively (about 80% recovery). EDTA presented a protective action on this recovery compared to the use of heparin as anticoagulant for snake blood. A reduction of vasotocin content related to the time of incubation of this peptide added to snake plasma was detected by radioimmunoassay. Snake plasma activity also on L-cystine-di-beta-naphthylamide indicated that this vasotocin-destroying effect was due to hydrolysis by a cystine-aminopeptidase-like activity. Plasma levels of vasotocin revealed an unexpected dispersion and absent correlation with plasma levels of osmolality. Measurable vasotocin in a large number of snakes associated with lower levels of l-cystine-di-beta-naphthylamidase in acute than in chronic salt loading suggested the role of this enzyme activity in long-term regulation of the vasotocin system in this snake.

Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper.

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23-25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23-25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I-IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

Intraerythrocytic inclusions associated with iridoviral infection in a fer de lance (Bothrops moojeni) snake.

Intraerythrocytic inclusions associated with infection by an iridovirus were observed in a fer de lance (Bothrops moojeni) snake that was being evaluated for the presence of renal carcinoma. The erythrocytes contained two types of inclusions, one viral and one crystalline, usually concomitantly. The snake was markedly anemic and exhibited a marked regenerative response. Ultrastructural analysis identified the virus to be an iridovirus consistent with snake erythrocyte virus and the crystalline structures to be of a different nature than hemoglobin.

Emponzoñamiento ofidico en pediátria años 1983-1993 H.P.E.C Valera / Snake poisoming in the pediatric years 1983-1993 H.P.E.C Valera

Con la finalidad de conocer las características clínicas, evaluar la eficacia del tratamiento del emponzoñamiento ofídico en la población pediátrica, se realizó un estudio retrospectivo en el cual se revisaron 69 casos de mordedura de serpiente, atendidos en la Emergencia Pediátrica del Hospital Central de Valera "Dr. Pedro Emilio Carrilo", desde enero de 1983 a diciembre de 1993. Las serpientes del género BOTHROPS fueron las responsables de los accidentes. Los hallazgos clínicos más frecuentes fueron: Edema, dolor, equimosis y hemorragias. Las mayores complicaciones se presentaron en 13 pacientes y de ésta la más frecuente fue la celulitis (61,5 por ciento). El emponzoñamiento ofídico se presentó en mayor proporción en el medio rural (88,4 por ciento) afectando especialmente al sexo masculino (55,1 por ciento). Los grupos etarios más afectados fueron los escolares (62,3 por ciento). Seguidos de los pre-escolares 37,7 por ciento. El 84 por ciento de las mordeduras ocurrió en los miembros inferiores, con predominio a nivel de pie (71 por ciento). La mortalidad representó el 0,69 por ciento. El 92,7 por ciento de los pacientes recibió tratamiento con suero antiofidio. El emponzoñamiento fue más frecuente en 1991 y la incidencia fue mayor en el mes de diciembre (17,4 por ciento)

Isolation and identification of angiotensin-like peptides from the plasma of the snake Bothrops jararaca.

Two distinct hypertensive peptides were purified and characterized from Bothrops jararaca (Bj) plasma incubated at pH 4, 37 degrees C, 24 hr. These peptides were active on rat and Bj blood pressure, on rat isolated uterus, on guinea pig isolated ileum and on Bj isolated duodenum. At the releasing conditions no further activities were found for kininases, angiotensinases or angiotensin converting enzymes. The peptides were purified by ethanol/ether extraction, Sephadex G-25 gel filtration, semipreparative reverse-phase (C-18) HPLC and analytical (C-18) HPLC. The amino-acid sequences of the purified peptides corresponded to (Ile5)AII and (Val5-Tyr9)AI and their molecular masses were confirmed by mass spectrometry as 1046.6 and 1348.0 respectively. The presence of those two angiotensins on Bj plasma may have some evolutionary significance since (Ile5)AII is known as a mammalian angiotensin and (Val5)AII as a non-mammalian one.

Isolation, characterization and mode of neutralization of a potent antihemorrhagic factor from the serum of the snake Bothrops asper.

A potent antihemorrhagic factor (BaSAH1) was isolated from the serum of the snake Bothrops asper by ammonium sulfate precipitation at 40-60%, Sephacryl S-200 and Sephadex G-50 gel filtration, DEAE-Sepharose, and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 5.2 and a molecular weight of 66 kDa. 4 micrograms of the purified factor BaSAH were needed to neutralize the hemorrhagic dose of B. asper whole venom compared to 60 micrograms of the clinically used horse polyvalent immunoglobulins. Moreover, 0.35 microgram of BaSAH were sufficient to achieve complete neutralization of the main hemorrhagic toxin (BaH1), with a molar ratio of 2:1. The antihemorrhagic activity was stable between pH 1.5-9 and up to 60 degrees C but lost activity completely after 30 min of heating at 70 degrees C. BaSAH did not digest the hemorrhagic toxin BaH1 or formed a precipitin line with it, nor with the whole venom. Both ELISA experiments and chromatography of BaSAH after incubation with the 125I-labeled hemorrhagic toxin BaH1 demonstrated that the mechanism of the neutralization involves a formation of an inactive soluble complex between the natural antihemorrhagin and the main hemorrhagin of B. asper venom.