Cysteine proteinase inhibitors in elapid and hydrophiid snake venoms.

The ability of elapid and hydrophiid snake venoms to inhibit cathepsin L was tested. All nine species of elapid and three species of hydrophiid snake venoms tested showed inhibition against cathepsin L. All of these venoms tested also showed inhibition against papain as well as against cathepsin L. Among these venoms, two elapid (Laticauda semifasciata venom, and Ophiophagus hannah venom) and one hydrophiid snake venom (Notechis scutatus scutatus venom) showed strong inhibition against both cathepsin L and papain. These venoms contained 12.0-13.0 kDa low molecular-weight cysteine proteinase inhibitors.     

The effects of Western Diamondback Rattlesnake (Crotalus atrox) venom on the production of antihemorrhagins and/or antibodies in the Virginia opossum (Didelphis virginiana).

Opossums are animals that are naturally resistant to the proteolytic effects of Crotalid venoms. Opossums possess proteinase inhibitors in their sera that bind to and neutralize hemorrhagic and other proteolytic activity in many snake venoms. The proteinase inhibitors are not antibodies since they have different molecular weights (60kDa) and pI (4.2). The purpose of this study was to determine if opossums were capable of producing antibodies against venom and/or increasing the production of proteinase inhibitors (specifically antihemorrhagins). Five different venom immunization protocols were used to determine the effects of the venom in the opossums. The dosages ranged from 1mg of venom per immunization to 350mg/kg body weight of venom per immunization. The antihemorrhagic response was increased, but there is no evidence to suggest that an opossum can produce antibodies against venom. The lack of an antibody response is most likely due to the natural proteinase inhibitors clearing the venom from the opossum's body before an antibody response can occur.     

Phospholipase A and B activities of reptile and Hymenoptera venoms

Phospholipase B (PLB) activity was found in a variety of snake, Gila monster and Hymenoptera venoms. Hymenoptera venoms, except bee, are an especially rich source of PLB. This was shown by incubation of crude venoms with lysophosphatidylcholine and subsequent titration of liberated fatty acids. PLB activity was not found in pure - or β-bungarotoxin, -cobrotoxin, or one crude snake venom (Crotalus horridus horridus). Snake, Gila monster and bee venoms exhibit higher PLB activity at an alkaline pH, while hornet venom PLB activity is greater at a neutral pH. Phospholipase A₂ (PLA₂) activity was determined using phosphatidylcholine and egg yolk as substrates. In reptile and bee venoms PLA₂ activity is much higher than PLB activity. In contrast, hornet venom PLA₂ and PLB activities are nearly equal. In reptile venoms PLA₂ activity is optimal at neutral pH, in contrast to PLB activity, suggesting that separate proteins or active sites are responsible for PLA₂ and PLB activities. The effect of boiling or heating venoms in either an acidic or alkaline milieu upon subsequent PLA₂ and PLB activity, measured at 37°C, was examined. The PLA₂ and PLB activities of all venoms tested were lost upon boiling at pH 9.4, except for the PLA₂ activity of Notechis scutatus venom which retained about 30% of its activity. Boiling at pH 5.5 results in greatly varying extents of retention of PLA₂ and PLB activities, dependent upon the venom examined. Therefore, the heat stability characteristics for each venom must be experimentally determined, not assumed. Boiling destroys PLA₂ and PLB activities of oriental hornet venom at about the same rate. No conclusive results were obtained from the heating studies as to whether PLA₂ and PLB activities reside upon the same molecule. However, PLA₂ and PLB activities of oriental hornet venom were separated using triple tandem column gel permeation chromatography, demonstrating the existence of two separate proteins for these activities.     

Comparison of two methods for proteolytic enzyme detection in snake venom

An acrylamide gel system containing fibrinogen was used to detect proteolytic enzymes in snake venoms. Proteolytic activity was observed as a clear area on a blue background after electrophoresis and overnight incubation in Tris buffer, prior to staining with Coomassie blue. Venoms from eastern and western diamondback and west coast Mexican rattlesnakes, Crotalus adamanteus, C. atrox and C. basiliscus basiliscus, respectively, and southern copperhead, Agkistrodon contortrix contortrix, were analyzed at the level of 1 mg of venom. The effect of the serine proteinase inhibitor diisopropylfluorophosphate (DFP) and the metalloproteinase inhibitors tetraethylenepentamine (TEP) or EDTA on fibrinogen and normal gel profiles were evaluated. Normal gels (without fibrinogen) were Coomassie stained to visualize migration of 250 micrograms of venom proteins on the gels. Several proteolytic enzymes detected in C. atrox and C. b. basiliscus venoms were inhibited by TEP, whereas DFP had no effect on activity. The fibrinogen gels detected no protease activity in C. adamanteus venom, although it is known from other studies that there are several proteolytic enzymes in this venom. Several proteases were detected in A. c. contortrix venom, one of which was inhibited by TEP. By comparison, proteolytic activity in 5-10 micrograms of all venoms was readily detected using the mammalian plasma kallikrein specific chromogenic substrate, S2302 (H-D-Pro-Phe-Arg-p-nitroanilide). The fibrinogen gel method does not appear to have the specificity nor the sensitivity of the recently developed chromogenic substrates for detection of proteolytic enzymes in snake venom.     

Proteolytic, hemorrhagic and hemolytic activities of snake venoms

Proteolytic, hemorrhagic and hemolytic activities were tested on 47 different venoms from the Crotalidae, Viperidae, Elapidae, and Hydrophiidae families. Antihemorrhagic activity of crude opossum (Didelphis virginiana) and woodrat (Neotoma micropus) serum was tested against the venoms that presented hemorrhagic activity. All venoms showed proteolytic activity when non-specific substrates such as hide powder and collagen were used. Members of the Crotalidae family had the highest hide powder, chymotrypsin-like and hemorrhagic activity. However, members of the Elapidae family had the highest collagen activity. Hemolytic activity was present in 85% of the snake venoms tested. The crude opossum and woodrat serum neutralized the hemorrhagic activity of all the hemorrhagic venoms. Of particular interest is the poor correlation between the venom activities measured here and the phylogenetic position of the snake that possess them. This is particularly true at the genus and species level. Differences in activities were found among individuals of the same genus. The significance of these differences among venoms of closely related snakes is unknown. They do not seem to be adaptive, however little is known of the physiology and habits of most venomous snakes.     

Extremely low nerve growth facior (NGF) activity of sea snake (Hydrophiidae) venoms

Sea snake venoms contain less protein than those of land snakes (Toom et al., 1969). Sea snake venoms lack arginine ester hydrolyzing activity, whereas those of Crotalidae and Viperidae have such activity (Tu et al., 1966). Sea snakes live in salty water, and their venoms may be different from those of land snakes. Because of the difficulty in obtaining sea snake venoms, information about sea snake venoms is quite incomplete. NGF is commonly present in the venoms of land snakes such as Elapidae, Viperidae, and Crotalidae (Cohen and Levi-Montalcini, 1956; Lipps, 2002). It is therefore of interest to investigate the presence or absence of NGF in sea snake venoms. In order to investigate the presence or absence of NGF, five sea snake venoms were selected. Lapemis hardwickii (Hardwick's sea snake) and Acalyptophis peronii venom were obtained from the Gulf of Thailand. Hydrophis cyanocinctus (common sea snake) and Enhydrina schistosa (beaked sea snake) venom were obtained from the Strait of Malacca. Laticauda semifasciata (broad band blue sea snake) venom was also examined and the venom was obtained from Gato Island in the Philippines.     

Characterization of a metallo-proteinase from Bothrops asper (terciopelo) snake venom

Metalloproteinase from the venom of Bothrops asper (proteinase G) is a glycoprotein with 1% neutral hexose and 3.5 moles of sialic acid per mole of protein. It hydrolyses a number of protein substrates such as casein, hemoglobin, gelatin and fibrinogen, whose alpha chain is degraded preferentially. The pH optimum of hydrolysis of casein is approximately 9.5. The protease is devoid of hemorraghic, esterolytic and amidolytic activities. The proteolytic activity of the enzyme increases by about 20% in the presence of 0.2 mM Ca2+ and Mg2+. Among the other ions tested, only Cd2+ and Fe2+ markedly decreased its activity. EDTA and cysteine are also strong inhibitors. In the presence of Ca2+ and EDTA, Zn2+ ions restored 50% of the activity. The amino acid composition shows fewer acidic residues than in related proteinases from other snake venoms.     

Variation in venom composition and reactivity in two specimens of yellow-faced whip snake (Demansia psammophis) from the same geographical area

Gel filtration chromatography and SDS-PAGE of venom from two specimens of Demansia psammophis showed little similarity. Amidolytic activity of the venoms, however, was in the same order of reactivity against various chromogenic substrates. The venom from both snakes produced precipitin lines with brown snake antivenom but the venom detection kit (Commonwealth Serum Laboratories) identified one venom as brown snake (Pseudonaja sp.) and the other as tiger snake (Notechis sp.). These results raise questions about the phylogeny of this species.     

Neutralization of venoms from two Southern Pacific Rattlesnakes (Crotalus helleri) with commercial antivenoms and endothermic animal sera.

The Southern Pacific Rattlesnake (Crotalus helleri) is found in southwestern California (USA), southward through north Baja California (MX) into the northern part of southern Baja California (MX). In this study, the venoms from two Southern Pacific Rattlesnakes were characterized. The two venoms were different in color, concentration, and enzyme activities. Two commercial antivenoms neutralized both C. helleri venoms differently. Antivipmyn (Fab2H) and CroFab (FabO) neutralized both venoms but had different ED50. Four times more Fab2H antivenom was required to neutralize the C. helleri venom No. 011-084-009 than the venom from the snake No. 010-367-284. The hemorrhagic activity of two C. helleri venoms were neutralized differently by endothermic animal sera having a natural resistance to hemorrhagic activity of snake venoms. Opossums and Mexican ground squirrel sera did not neutralize the hemorrhagic activity of the venom No. 010-367-284. The sera of gray woodrats and hispid cotton rats neutralized all hemorrhagins in both C. helleri venoms. This is the first reported case in which opossum serum has not neutralized hemorrhagic activity of pit viper venom. Differences in the compositions of C. helleri venoms and their ability to be neutralized may help explain why snakebites are a difficult medical problem to treat and why effective polyvalent antivenoms are difficult to produce.     

Citrate inhibition of snake venom proteases

Thirty snake venoms had a citrate content of 2.3 to 12.9%, dry basis, by an aconitase–isocitric dehydrogenase coupled enzyme assay. This is a venom concentration range of approximately 30 to 150 mM citrate assuming 25% venom solids content. Inhibition of snake venom protease activity by the addition of exogenous citrate was obtained using azure blue hide powder and azocasein as substrates. Protease inhibitions of 7.5% for Crotalus atrox venom to 78% for Bothrops picadoi venom were observed with citrate. Complete inhibition of snake venom protease activity by citrate was not observed. Bothrops asper (Pacifico) venom showed a 41% protease inhibition by citrate with azocasein as the substrate and 46% inhibition of Bothrops asper (Alantico) venom protease with azure blue hide power as a substrate. Trypsin was not inhibited in this system. Citrate may inhibit some venom protease activity by forming a complex with the zinc of zinc-dependent enzymes.     

Cross neutralization of dangerous snake venoms from Africa and the Middle East using the VACSERA polyvalent antivenom. Egyptian Organization for Biological Products & Vaccines

This study was performed to assess the ability of polyvalent snake venom anti-serum, produced by the Egyptian Organization for Biological Products & Vaccines (VACSERA), to neutralize several toxic activities of snake venoms, not only of those included in the antivenom mixture, but also some additional venoms of snakes from Egyptian, African, and Middle Eastern habitats. In general, the results revealed that polyvalent snake venom anti-serum from VACSERA is highly effective in neutralizing Egyptian snake venoms, especially Naja haje, Naja nigricolles, Naja pallida, Cerastes cerastes, Cerastes cerastes cerastes, Cerastes vipera, Pseudocerastes persicus fieldi, and Walterinnisia egyptia. The antivenom was also effective against Naja haje, Walterinnisia egyptia, and Bites aritans from Saudi Arabia. High activity was obtained against venoms from Naja haje, Naja nigricolles, and Naja pallida of Sudan, as well as the African Naja melanoleuca, Naja mossambica, Naja naja oxiana, Bites gabonica, and Vipera lebetina. Only moderate effectiveness was obtained with Echis coloratus and Echis carinatus, and the polyvalent antiserum was ineffective against the venom of Naja nivea.     

Snake venom: kill and cure

Snake venom is a natural biological resource that contains several components, which are not only responsible for death but also have a potential therapeutic activity. The use of snake venom for medicinal purposes dates back to ancient times, now many drugs and clinical diagnostic kits have derived from components of snake venom. The scientists can extract, purify and identify new components of venom that may serve as starting point for structure–function relationship studies leading to design new medications. This review will highlight the activities of snake venoms and their components against cancer, microbes, parasitic infections and platelet aggregation.     

Capacity of Thai green pit viper antivenom to neutralize the venoms of Thai Trimeresurus snakes and comparison of biological activities of these venoms

The capacity of Thai green pit viper antivenom raised to Trimeresurus albolabris to neutralize the venoms from six species of Trimeresurus sp. in Thailand has been examined. They were Trimeresurus albolabris, T. macrops, T. popeiorum, T. hageni, T. purpureomaculatus, and T. kanburiensis. The antivenom neutralized lethal and hemorrhagic activities of all these venoms. The capacity of antivenom to neutralize lethal toxicity of the venom was expressed as the amounts (mg) of snake venom neutralized by 1 ml of the antivenom. The largest capacity was found with the homologous venom. Results of immunodiffusion, immunoblotting, and antigen-antibody complex formation experiments supported the results of neutralization experiments. Several biological activities of the Trimeresurus venoms were also examined and compared. They were lethal, hemorrhagic, proteolytic, phospholipase A, arginine ester hydrolyse, and thrombin activities. There was no correlation between the ratios of lethal toxicity and hemorrhagic activity, lethal toxicity and phospholipase A activity, as well as hemorrhagic activity and proteolytic activity.     

Snake venoms and the hemostatic system

Snake venoms are complex mixtures containing many different biologically active proteins and peptides. A number of these proteins interact with components of the human hemostatic system. This review is focused on those venom constituents which affect the blood coagulation pathway, endothelial cells, and platelets. Only highly purified and well characterized snake venom proteins will be discussed in this review. Hemostatically active components are distributed widely in the venom of many different snake species, particularly from pit viper, viper and elapid venoms. The venom components can be grouped into a number of different categories depending on their hemostatic action. The following groups are discussed in this review: (i) enzymes that clot fibrinogen; (ii) enzymes that degrade fibrin(ogen); (iii) plasminogen activators; (iv) prothrombin activators; (v) factor V activators; (vi) factor X activators; (vii) anticoagulant activities including inhibitors of prothrombinase complex formation, inhibitors of thrombin, phospholipases, and protein C activators; (viii) enzymes with hemorrhagic activity; (ix) enzymes that degrade plasma serine proteinase inhibitors; (x) platelet aggregation inducers including direct acting enzymes, direct acting non-enzymatic components, and agents that require a cofactor; (xi) platelet aggregation inhibitors including: -fibrinogenases, 5′-nucleotidases, phospholipases, and disintegrins. Although many snake venoms contain a number of hemostatically active components, it is safe to say that no single venom contains all the hemostatically active components described here. Several venom enzymes have been used clinically as anticoagulants and other venom components are being used in pre-clinical research to examine their possible therapeutic potential. The disintegrins are an interesting group of peptides that contain a cell adhesion recognition motif, Arg–Gly–Asp (RGD), in the carboxy-terminal half of their amino acid sequence. These agents act as fibrinogen receptor (integrin GPIIb/IIIa) antagonists. Since this integrin is believed to serve as the final common pathway leading to the formation of platelet–platelet bridges and platelet aggregation, blockage of this integrin leads to inhibition of platelet aggregation regardless of the stimulating agent. Clinical trials suggest that platelet GPIIb/IIIa blockade is an effective therapy for the thrombotic events and restenosis frequently accompanying cardiovascular and cerebrovascular disease. Therefore, because of their clinical potential, a large number of disintegrins have been isolated and characterized.     

Pharmacologic and enzymatic effects of snake venoms from Antioquia and Choco (Colombia)]

We compared several pharmacological and enzymatic effects induced by 11 snake venoms from seven species, six of them from different geographic areas of Antioquia and Choco, north-west of Colombia, South America (Bothrops atrox, B. nasutus, B. schlegelii, B. punctatus, Lachesis muta, Micrurus mipartitus), and Crotalus durissus terrificus venom, from specimens captured in other provinces of the country (Tolima, Huila, Meta and Atlantico). Differences were observed in edema-forming, hemorrhage, defibrination, indirect hemolysis, myonecrosis, proteolysis and lethal activity between venoms from different genera or species, as well as according to the geographic area of origin in B. atrox and B. nasutus snake venoms. Bothrops venoms, in particular B. atrox and L. muta, produced major local effects. All of the venoms, including M. mipartitus, had myotoxic effects. The most defibrinating venoms were B. atrox, L. muta, B. punctatus and C. d. terrificus. All of the venoms had indirect hemolytic activity; the venom of M. mipartitus being greatest. The most lethal venoms were those of C. d. terrificus and M. mipartitus. Within Bothrops species, the venom of B. schlegelii was the least active in terms of local and systemic pathologic effects.     

A new method for the detection of phospholipase A2 variants: identification of isozymes in the venoms of newborn and adult Bothrops asper (terciopelo) snakes

A new method for the identification of phospholipase A2 isozymes in snake venoms is described. The technique is based on the separation of the venom components by isoelectric focusing in agarose gels, transfer of the protein bands by diffusion onto nitrocellulose paper and detection of the phospholipolytic activity of the enzymes by a hemolytic assay either in agarose gels or by benzidine reaction on a solid matrix. Striking differences in the electrophoretic patterns of the phospholipase A2 isozymes between the Atlantic and Pacific venoms and between the newborn and adult venoms from Bothrops asper specimens were observed. The method allowed the detection of 9 different phospholipase A2 isozymes in the venom of adult Atlantic, 7 isozymes in the venom of adult Pacific, and 2-3 isozymes in the venoms of newborn specimens. Horse polyvalent antivenom varied in its capacity to neutralize the phospholipolytic activity of the different isozymes in the same venom and among different venoms.     

Comparative study of nine Bothrops snake venoms from adult female snakes and their offspring.

Venoms of seven different Bothrops species and three subspecies (B. alternatus, B. cotiara, B. erythromelas, B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi paranaensis. B.n. pauloensis and B.n. urutu) obtained from individual mothers and their young were investigated. Biometrics of snakes and protein content, toxicity (LD50), SDS-PAGE, proteolytic and clotting activities of venoms were estimated. Comparison of venoms from female snakes and their respective newborn offspring were variable in protein content, toxicity, fibrinolytic/amidolytic/thrombin-like activities and in venom yield in relation to snake length. B.n. paranaensis and B.n. pauloensis possessed the most toxic venoms. Caseinolytic activity of all venoms from female snakes and procoagulant activity of their offspring were consistently high. Venoms of B. erythromelas mother and offspring had no amidolytic activity and the highest levels of factor X and prothrombin activators without thrombin-like action. In contrast, the venom of newborn B. cotiara possessed the highest thrombin-like activity whereas a B. jararacussu adult female did not posses any procoagulant activity. An extremely high procoagulant activity of the venom of newborn Bothrops specimens was demonstrated.     

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms.

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes.