Differential action of proteases from Trimeresurus malabaricus, Naja naja and Daboia russellii venoms on hemostasis

The action of venom proteases and their role in hemostasis has been compared in the venoms of Trimeresurus malabaricus, Daboia russellii and Naja naja from the Southern region of Western Ghats, India. These venoms exhibit varying amounts of proteolytic activity and also influence hemostasis differently. Casein hydrolyzing activity of T. malabaricus venoms was 16 and 24 fold higher than those of N. naja and D. russellii venoms, respectively. With the synthetic substrate TAME, the highest activity was observed in T. malabaricus venom. N. naja venom did not hydrolyze TAME even at higher concentrations. These variations in proteolytic activity also influenced the coagulation process. T. malabaricus and D. russellii venoms are strongly procoagulant and reduce the re-calcification time from 148 to 14 and 12 s, respectively. Similarly, both T. malabaricus and D. russellii venoms reduce the prothrombin time from 12.5 to 6.0 s. On the other hand, N. naja venom is anticoagulant and prolongs re-calcification time to 600 s and prothrombin time to 42 s. In spite of varied effects on hemostasis, all the venoms hydrolyze fibrinogen. T. malabaricus venom hydrolyses both Aalpha and Bbeta subunits. While D. russellii and N. naja venoms hydrolyse only Aalpha. None of these venoms hydrolyze the gamma subunit of fibrinogen. Inhibition studies with specific protease inhibitors revealed that both N. naja and T. malabaricus venoms contain only metalloproteases. D. russellii venom contained both serine and metalloproteases. Only, T. malabaricus venom exhibited thrombin-like activity and induces fibrin clot formation with purified fibrinogen within 58 s. Even though D. russellii venom exhibits procoagulant activity, it did not show thrombin-like activity and may act on other coagulation factors.     

Biochemical composition, lethality and pathophysiology of venom from two cobras-- Naja naja and N. kaouthia

The cobras Naja naja and N. kaouthia are abundant in eastern and north-eastern India, accounting for maximum snakebite deaths. Here we report on variation in the composition of Naja kaouthia and N. naja venom from eastern India on corresponding differences in the severity of pathogenesis. These two venoms differ in chromatographic elution profile through Sephadex G-50 and enzyme activity, protein and carbohydrate contents associated with each fraction. The presence of greater amounts of basic phospholipase A2, L-amino acid oxidase and low molecular weight membrane active polypeptides in the N. naja venom makes it more toxic than N. kaouthia venom. A commercial polyvalent antivenom raised against N. naja venom inactivates lethality and variety of toxic effects of homologous venom more effectively than N. kaouthia venom.     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom (Apis mellifera), three snake venoms (Naja naja sputatrix, Vipera russellii and Crotalus adamanteus) and the polypeptide melittin was investigated against Escherichia coli. Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom greater than melittin greater than N. naja sputatrix venom much greater than V. russellii venom greater than C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom ( Apis mellifera ), three snake venoms ( Naja naja sputatrix, Vipera russellii and Crotalus adamanteus ) and the polypeptide melittin was investigated against Escherichia coli . Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom > melittin > N. naja sputatrix venom ≫ V. russellii venom > C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.     

Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle

The effects of cardiotoxin fractions from Naja naja kaouthia and Naja naja atra snake venoms and synthetic melittin peptide were examined on lipolytic activity in red blood cells and primary skeletal muscle cultures. Both native cardiotoxin fractions caused considerable production of free fatty acids in red blood cells. This production was abolished when the fractions were first treated with p-bromophenacyl bromide to reduce the venom phospholipase A2 activity contamination. In equine and human primary cultures of skeletal muscle, the N. n. kaouthia cardiotoxin (10 microM) and melittin (2 microM) caused a breakdown of phospholipids and production of free fatty acids and diacylglycerol in the absence of lysophospholipid formation. Additionally, melittin at higher concentrations (10 microM) caused triglyceride breakdown. These studies do not support the suggestion that snake venom cardiotoxins and melittin selectively activate endogenous phospholipase A2 activity. Instead, the toxins primarily activate endogenous phospholipase C activity and, in the case of melittin at high concentrations, triglyceride lipase activity.     

The anticoagulant activity of Malayan cobra (Naja naja sputatrix) venom and venom phospholipase A2 enzymes

Malayan cobra (Naja naja sputatrix) venom was found to exhibit an in vitro anticoagulant activity that was much stronger than most common cobra (genus Naja) venoms. The most potent anticoagulants of the venom are two lethal phospholipase A2 enzymes with pI's of 6.15 and 6.20, respectively. The anticoagulant activity of the venom is due to the synergistic effect of the venom phospholipase A2 enzymes and polypeptide anticoagulants. Bromophenacylation of the two phospholipase A2 enzymes reduced their enzymatic activity with a concomitant drop in both the lethal and anticoagulant activities.     

Lipid monolayers: interactions with the apoprotein of high density plasma lipoprotein

Monolayer techniques were used to study the interactions of various lipids (cholesterol, lysophosphatidyl choline, phosphatidal ethanolamine, phosphatidyl choline, sphingomyelin, stearic acid, and lipids extracted from plasma high density lipoproteins and very low density lipoprotein) with the lipid-free protein subunit of rat plasma high density lipoprotein and with rat plasma albumin. The proteins were injected under the lipid monolayer at fixed area, and the increase in surface pressure (decrease in surface tension) was measured as a function of time. With all lipids, both the rate and magnitude of this increase were greater with the apolipoprotein than with albumin. The degree of film penetration of pure lipid films (at an initial film pressure of 15 dynes/cm) by the two proteins followed the same order: cholesterol > phosphatidal ethanolamine > phosphatidyl choline > stearic acid > sphingomyelin > lysophosphatidyl choline. Other variables studied were protein concentration, initial film pressure, and pH. Two distinctive properties of the apolipoprotein were the penetration of lipid films at pressures above the collapse pressure of the protein, and the formation of a film even at low salt concentration. High surface activity and strong interaction of HDL-protein with lipid monolayers may be associated with the flexibility of the protein molecule due to absence of disulfide bridges. The unusual surface activity of HDL-protein may be intimately related to the mechanism of formation of the lipoprotein.     

Inactivation of phospholipase A2 and metalloproteinase from Crotalus atrox venom by direct current

To achieve our aim of understanding the interactions between direct current and enzymes in solution, we exposed reconstituted Crotalus atrox venom to direct electric current by immersing two platinum thread electrodes connected to a voltage generator (between 0 and 8 V) into a reaction mixture for a few seconds. Then, we assayed the residual activity of phospholipases A(2) (PLA(2)),metalloproteinases, and phosphodiesterases, abundant in crotaline snake venoms and relevant in the pathophysiology of envenomation, characterized by hemorrhage, pain, and tissue damage. C. atrox venom phospholipase A(2) and metalloproteinases were consistently and irreversibly inactivated by direct current (between 0 and 0.7 mA) exposure. In contrast, C. atrox venom phosphodiesterases were not affected. Total protein content and temperature of the sample remained the same. Secretory pancreatic phospholipase A(2), homologue to snake venom phospholipases A(2), was also inactivated by direct current treatment. In order to understand the structural reasoning behind PLA(2) inactivation, circular dichroism measurements were conducted on homogeneous commercial pancreatic phospholipase A(2), and it was found that the enzyme undergoes structural alterations upon direct current exposure.     

Suicide-inhibitory bifunctionally linked substrates (SIBLINKS) as phospholipase A2 inhibitors. Mechanistic implications

Novel phospholipids that function as mechanism-based inhibitors for phospholipase A2 (PLA2) are described. PLA2-catalyzed hydrolysis of the sn-2 ester of these suicide-inhibitory bifunctionally linked substrates (SIBLINKS) followed by a cyclization reaction generates a cyclic anhydride at the active site of the enzyme which leads to inhibition. Structure/activity relationships for the SIBLINKS substituents in the sn-1 and sn-2 position are delineated. Time courses and efficiency of SIBLINKS inhibition are reported and compared for extracellular PLA2s obtained from Naja naja naja, porcine pancreas, bee venom, Crotalus atrox and Crotalus adamanteus. SIBLINKS-inhibited PLA2s cannot process either monomeric or micellar substrates consistent with inhibition at the catalytic site. Some SIBLINKS efficiently inactivate 1 mol of N. naja naja and C. adamanteus PLA2/6-10 mol of SIBLINKS hydrolyzed. Inhibition of N. naja naja PLA2 can be reversed by hydroxylamine, suggesting that a tyrosine residue is acylated.     

Inactivation of Crotalus atrox venom hemorrhagic activity by direct current exposure using hens' egg assay

The hemotoxic venoms of Viperidae and Crotalidae are responsible for most of the evenomations in the United States, West Africa, India, South-East Asia, New Guinea, and Latin America. We previously reported that a short exposure of Crotalus atrox venom to direct electric current (dc) from a low-voltage generator, in solution, causes consistent and irreversible inactivation of venom phospholipase A(2) and metalloproteases. Here we report by in vivo assay on chicken embryos at stage 18 of development according to Hamburger and Hamilton that the hemorrhagic activity of C. atrox venom is lost after exposure to dc (from low voltage). Venom was exposed to dc ranging between 0 and 1 mA. dc values above 0.7 mA abolished hemorrhage. Such in vivo data, showing that dc neutralizes C. atrox venom hemorrhagic activity suggest that a deeper knowledge is needed to understand the relationship among dc and biological matter.     

Acid and alkaline phosphomonoesterase activities in snake venoms

• 1.1. Acid and alkaline phosphatase activities of eight different snake venoms were determined quantitatively by using synthetic substrates, o-carboxyphenylphosphate and p-nitrophenylphosphate respectively.
• 2.2. It was found that most of Elapidae venoms investigated had both acid and alkaline phosphatase activities.
• 3.3. Three Crotalidae venoms investigated did not show any alkaline phosphatase activity.
• 4.4. The strength of venom acid phosphatase activity is as follows: Agkistroden acutus > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.
• 5.5. The strength of venom alkaline phosphatase activity by using p-nitrophenylphosphate is in the order of Naja hannah > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.When o-carboxyphenylphosphate was used as a substrate, the order of enzyme activity is Naja hannah > Naja haje > Naja naja samarensis > Naja melanoleuca > Naja naja atra.
• 6.6. Acid phosphatase activity of all the Elapidae venoms was inhibited completely by fluoride. The alkaline phosphatase activity of Elapidae venoms was not inhibited by fluoride either using p-nitrophenylphosphate or o-carboxyphenylphosphate.
• 7.7. The acid phosphatase of all the Elapidae venoms was not inhibited by zinc ion. However, most of the venom alkaline phosphatases were inhibited by zinc ion.
• 8.8. Ethylenediaminetetraacetic acid (EDTA) had inhibitory action on venom phosphatase activity. However, tris-(hydroxymethyl)-aminoethane had a counter effect on the inhibitory action of EDTA.
• 9.9. Optimum pH studies of the snake venom phosphatases showed that the acid phosphatases of the snake venoms had their highest activity in the range of pH 4–5. The alkaline phosphatases of the snake venoms had their optimum pH at 9.
• 10.10. Comparable experiments were also conducted by using chicken intestine alkaline phosphatase and wheat germ acid phosphatase.

     

Effects of pH and calcium ion on self-association properties of two dimeric phospholipases A2

The monomer-dimer equilibria of the dimeric phospholipases A2 from Crotalus atrox and Agkistrodon piscivorus piscivorus venoms were examined chromatographically as a function of pH and in the presence versus absence of the essential cofactor, calcium ion. At neutral pH without calcium, the subunits of both enzymes reequilibrated sufficiently slowly that dimer and monomer were separated by size exclusion chromatography. At pH 4.2 and lower, the dimers underwent rapid dissociation and reassociation, eluting as single broad peaks whose position as a function of applied protein concentration could be analyzed to determine association constants using an algorithm that estimates these values based on elution positions. Lowering the pH from 7.0 to 4.2 increased the self-association constant of the C. atrox enzyme by 1 order of magnitude and that of the A. p. piscivorus dimer by a factor of 3. Calcium ion, an essential cofactor of phospholipase A2, converted the kinetic behavior of the dimers at neutral pH from slow to virtually instantaneous on the time scale of the chromatography runs, 40 min. Calcium ion also altered the thermodynamic stability of the enzymes; the association constant of A. p. piscivorus phospholipase A2 in neutral pH buffer was reduced by approximately 2 orders of magnitude, whereas that of C. atrox was increased by a factor of 6. The structural basis for the disparate effects of calcium ion on these two acidic, dimeric venom phospholipases A2 is uncertain. This study illustrates the importance of calcium ion and pH on the solution behavior of the dimeric members of this class of enzymes.     

Variation in biochemical and pharmacological properties of Indian cobra (Naja naja naja) venom due to geographical distribution

Indian cobra (Naja naja naja) venom obtained from three different geographical regions was studied in terms of electrophoretic pattern, biochemical and pharmacological activities. SDS-PAGE banding pattern revealed significant variation in the protein constituents of the three regional venoms. The eastern venom showed highest indirect hemolysis and hyaluronidase activity. In contrast, western and southern venoms were rich in proteolytic activity. All the three regional venoms were devoid of p-tosyl-L-arginine methyl ester hydrolysing activity. The eastern venom was found to be most lethal among the three regional venoms. The lethal potency varied as eastern > western > southern regional venoms. In addition, all the three regional venoms showed marked variations in their ability to induce symptoms/signs of neurotoxicity, myotoxicity, edema and effect on plasma coagulation process. Polyclonal antiserum prepared against the venom of eastern region cross-reacted with both southern and western regional venoms, but varied in the extent of cross-reactivity by ouchterlony immunodiffusion and ELISA.     

Phospholipases: melittin facilitation of bee venom phospholipase A2-catalyzed hydrolysis of unsonicated lecithin liposomes.

Melittin isolated from the venom of the common honey bee is a potent activator for bee venom phospholipase A₂-catalyzed hydrolysis of unsonicated liposomes of egg phosphatidyl choline. At 37 °C and pH 8, the rate of this enzymatic reaction is increased approximately 300-fold by the addition of 8 × 10^?5m melittin. The magnitude of facilitation of the phospholipase A₂ reaction is much greater than that previously reported by other workers for systems involving sonicated egg phosphatidyl choline liposomes or Escherichia coli membrane fragments as substrates. Melittin having lysines quantitatively modified through reaction with methyl acetimidate is as effective a potentiator of phospholipase A₂ activity as the unmodified material. The same result was obtained for melittin in which the single tryptophan residue was modified. Melittin modified by succinylation retained approximately 50% of its capacity to facilitate phospholipase A₂ activity. In contrast, a modified melittin in which the C-terminal four amino residues were removed, acetimidated des(23–26)melittin, is a very poor activator, as is a mixture of this peptide with the C-terminal tetrapeptide. In contrast to the results with egg lecithin liposomes, melittin has little influence on the susceptibility of monomolecular aqueous solutions of dihexanoylphosphatidyl choline to phospholipase A₂ attack.     

Phosphatidyl choline interaction with bovine serum albumin: effect of the physical state of the lipid on protein-lipid complex formation.

Radioactively labeled [¹⁴C]phosphatidyl choline dispersed on Celite was equilibrated with bovine serum albumin solutions buffered at pH 8.0. Phosphatidyl choline was rapidly solubilized in the presence of serum albumin, and formed stable protein-lipid complexes which were isolated by gel-filtration through a Sepharose 4B column. Under similar conditions, equilibration of the protein with phosphatidyl choline liposome dispersions in buffer did not result in complex formation. The altered physical state of phosphatidyl choline on the weakly adsorbing Celite surface appears to be essential for binding by native bovine serum albumin. This work reports the first observation of phosphatidyl choline binding to native serum albumin in bulk phase and suggests the possibility of exposing monodisperse lipids, under controlled conditions, to proteins having lipid binding properties.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

眼镜蛇毒对大鼠脊髓和脊神经节一氧化氮合酶表达的影响

目的 观察眼镜蛇毒对脊髓和脊神经节一氧化氮合酶(NOS)表达的影响。方法 将眼镜蛇毒注入大鼠右侧大腿后部,采用还原型尼克酰胺嘌呤二核苷酸脱氢酶(NADPH．d)法显示NOS的表达。结果 在眼镜蛇毒注射组,脊髓和脊神经节内的NOS阳性神经元和深染NOS阳性神经元明显多于注入生理盐水组和正常对照组。结论 注入眼镜蛇毒能上调大鼠脊髓和脊神经节NOS表达。     

Proteolytic activities of cobra venoms based on inactivation of alpha 2-macroglobulin

The venoms of various cobra species showed a wide range of abilities to cleave hide powder azure, with Naja naja kaouthia and Ophiophagus hannah venoms showing the lowest activities and Naja nivea venom showing the greatest activity on this dye-linked substrate. The activities of the venoms on hide powder did not completely correlate with their ability to inactivate the alpha 2-macroglobulin of human serum. Incubation of 4-5 micrograms of Naja nigricollis venom per microliter of serum for 30 min caused loss of 95% of the alpha 2-macroglobulin activity of the serum. The inactivation was rapid, reaching 80% inactivation 5 min after mixing. This loss of alpha 2-macroglobulin activity was used to quantitate the weak proteolytic activity of N. nigricollis venom and a partially purified sample of the major fibrinogenolytic proteinase of the venom. The inactivation of alpha 2-macroglobulin was also used to compare the proteinase activities of venoms from seven species or subspecies of cobra. Based on alpha 2-macroglobulin inactivation, N. nigricollis had the highest proteinase activity among the tested venoms. The measurement of alpha 2-macroglobulin inactivation should provide a useful alternative to hide powder digestion for demonstration of weak proteolytic activities in venoms.     

Inhibitory effects on phospholipase A2 and antivenin activity of melanin extracted from Thea sinensis Linn

Antivenin activity of melanin extracted from black tea (MEBT) was reported for the first time. The antagonistic effect of MEBT was evaluated for Agkistrodon contortrix laticinctus (broadbanded copperhead), Agkistrodon halys blomhoffii (Japanese mamushi), and Crotalus atrox (western diamondback rattlesnake) snake venoms administered i.p. to ICR mice. MEBT was injected i.p. immediately after the venom administration in dose of 3 mg per mouse in the same place of venom injection. MEBT demonstrated neutralization effect against all venoms tested. The greatest antivenin effect of MEBT was found against Japanese mamushi snake venom. In this case, half the mice died within 2.5 +/- 0.7 h after injection of 0.9 mg/kg of venom. An immediate injection of MEBT substantially reduced the toxic effect of venom and extended time at the 50% level of survival up to 52.3 +/- 2.3 h. The antivenin activity of MEBT is due to chelating of Ca++ and non-specific binding of phospholipase A2. The inhibitory effect of MEBT on phospholipase A2 assessed for different venoms was similar to that obtained with pure enzyme. Low toxicity of MEBT in combination with its antagonistic activity against different venoms may allow effective life-saving treatment against snakebites. Such application of MEBT is important when identification of the snake is impossible or if specific treatment is unavailable.     

The composition of Naja naja venom samples from three districts of West Bengal, India

The variation in the composition of Naja naja venoms from three neighbouring districts of West Bengal, eastern India and the corresponding differences in the severity of pathogenesis due to venom composition variation are reported. These venom samples differ with respect to chromatographic elution profile and enzyme activity associated with each fraction. Presence of higher quantities of basic phospholipase and plasma protein hydrolase in the venom samples of Burdwan and Purulia make them more toxic than Midnapur venom sample. A polyvalent antivenom manufactured in western India was hardly effective in neutralizing the pathobiological manifestation of the venom samples from eastern India.