Venom Fractions Research Articles

Partial Fractionation of Venoms from Two Iranian Vipers, Echis carinatus and Cerastes persicus Fieldi and Evaluation of Their Antiplatelet Activity.

Platelet aggregation inhibitory effect and anticoagulant properties of fractions separated from the venoms of Cerastes persicus fieldi and Echis carinatus were investigated. The partial fractionation was performed on a Sephadex G-100 column. Two fractions separated from Cerastes persicus fieldi showed anti platelet aggregation activity on ADP (200 μM)-induced platelet aggregation (ca 80% inhibition). Attempts to measure the antiplatelet aggregation activity of crude Echis carinatus venom and its fractions were not successful due to the protein coagulation of the plasma samples after the addition of venom. Anticoagulant activities of venoms were also evaluated. Total venom of Echis carinatus showed anti coagulant activity in PT test, while its fractions showed procoagulant activity.

Isolation of a cytolytic component from <i>naja nigricollis nigricollis</i> (spitting cobra) venom*

A cytolytic compound (VIb) was isolated by fractionation of crude venom from Egyptian Cobra Snake, Naja nigricollis nigricollis using Sephadex G-75, followed by CM- Sephadex. The isolated protein has a cytolytic effect on red cells, stable at different temperatures (25 ºC-100ºC) and at different pH ranges (5-9). It is partially pure as indicated by SDS-PAGE, with molecular weight approximately 10 kDa. It has no protease or phospholipase activity. It is a glycoprotein, with maximum absorbance at 280nm. It is non antigenic, with LD50 1.83 mg/ kg body weight. DNA extraction from mouse liver tissue and from hepatocellular carcinoma cell line (HEPG2) gave a single intact band after invitro incubation with VIb, indicating that apoptosis is not involved in the cytotoxic mechanism. Small doses of VIb induced lysis in breast, colon and hepatocellular carcinomas cell lines in tissue culture plates.Keywords: Naja cytotoxin, Hemolytic, Cytolysis, malignant cells

Effect of toxic fraction of Hemiscorpius lepturus's venom in serum metabolome profile by the help of 1H- nuclear magnetic resonance

Effect of toxic fraction of Hemiscorpius lepturus's venom in serum metabolome profile by the help of 1H- nuclear magnetic resonance

Some pharmacological properties of the venom, venom fractions and pure toxin of the yellow-bellied sea snake Pelamis platurus

The effects of the crude venom, four partially purified venom fractions and pure toxin (Pelamis toxin alpha) from yellow-bellied sea snake, Pelamis platurus, on respiration, blood pressure, heart and skeletal muscle of rabbits have been examined. Results indicated that crude venom, a partially purified toxic fraction and Pelamis toxin alpha caused initial respiratory stimulant effects followed by respiratory paralysis. In most cases, respiratory paralysis occurred before a profound fall in arterial pressure. Depression of the twitch response to nerve stimulation was observed in the tibialis anterior muscle. No significant change in the electrocardiogram was seen. Three partially purified non-toxic fractions of the crude venom induced transient respiratory stimulant effects. It was concluded that the crude venom and Pelamis toxin alpha had an identical mode of action and that they caused respiratory paralysis in rabbits.

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard ( Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard ( Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster ( H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe) 5-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides.

Bosentan, a selective and more potent antagonist for Atractaspis envenomation than the specific antivenom

Preparation of an antivenom against Atractaspis had been done for the first time in the year 2007 in NAVPC in Riyadh, however, it was a lengthy and expensive process. In this study, an alternative treatment was tested using: 1- Nitroglycerin to antagonize the coronary vasospasm induced by the venom, 2- Bosentan to block the endothelin receptors since there is a similarity in structure and effect between the toxic fraction of venom (Sarafotoxins) and endothelins and 3- The specific Antivenom in comparison to nitroglycerin and bosentan. Pretreatment of rabbits with nitroglycerin, antivenom or bosentan completely protected all rabbits from the minimal lethal doses of venom or its toxic fraction. On the other hand, injecting any of the three drugs a few minutes (min) after injecting one minimal lethal dose (MLD) of the venom or the toxic fraction and at the first signs of ischemia, just before death, showed that bosentan completely saved all rabbits. In case of nitroglycerin all rabbits died and in case of antivenom, only 5 out of 10 rabbits were rescued. It is clear that bosentan is superior to the specific antivenom in protecting rabbits; this may be due to its higher affinity to endothelin receptors than sarafotoxins. This preclinical study shows a good potential in using bosentan as a selective antidote for atractaspis envenomation, especially in the African continent.

Effects of Bothrops moojeni venom on Leishmania amazonensis promastigote forms

Abstract: In the present work, the effect of Bothrops moojeni venom fractions on Leishmania promastigotes was evaluated. The snake venom was fractionated into five protein fractions (E1 to E5), by ion exclusion chromatography, that were used to treat Leishmania amazonensis and Leishmania braziliensis promastigote forms whereas the viability and nitric oxide production were evaluated. It was observed that E5 venom fraction strongly inhibited Leishmania amazonensis nitric oxide production, while in Leishmania braziliensis the nitric oxide production was enhanced in all doses. Bothrops moojeni crude venom reduced the viability of both parasites in a dose-dependent manner and a peptide of 64 kDa was apparently degraded. Bothrops moojeni E5 venom fraction only reduced the viability and nitric oxide production of Leishmania amazonensis and no protein degradation was observed. Thus, these results suggest that Bothrops moojeni E5 venom fraction may offer components with a promising antileishmanial therapeutic application.Key words:

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.

Enzymatic and toxinological activities of Hypnale hypnale (hump-nosed pit viper) venom and its fractionation by ion exchange high performance liquid chromatography

Hypnale hypnale (hump-nosed pit viper) has been recently identified as one of the medically important venomous snakes in Sri Lanka and on the southwestern coast of India. The characterization of its venom is essential for understanding the pathophysiology of envenomation and for optimizing its management. In the present study, the biological properties of Hypnale hypnale venom and venom fractions obtained using Resource Q ion exchange chromatography were determined. The venom exhibited toxic activities typical of pit viper venom, comparable to that of its sister taxon, the Malayan pit viper (Calloselasma rhodostoma). Particularly noteworthy were its high activities of thrombin-like enzyme, proteases, phospholipase A2, L-amino acid oxidase and hyaluronidase. The thrombin-like enzyme was mainly acidic and distributed over several chromatography fractions, indicating its existence in multiple isoforms. The hemorrhagic and necrotic activities of the venom were likely associated with the proteolytic enzyme found mainly in the basic fraction. Phospholipase A2 and phosphomonoesterase exist in both acidic and basic isoforms, while L-amino acid oxidase and hyaluronidase are highly acidic. The venom clotting activity on fibrinogens showed distinct species specificity in the following increasing order for clotting time: bovine < rabbit < goat < human < horse < < dog, and was comparable to that of C. rhodostoma venom. Its clot formation on human fibrinogen is gradual and prolonged, a phenomenon suggestive of consumptive coagulopathy as a complication observed clinically. At an intramuscular sublethal dose, the venom did not cause acute kidney injury in a rodent model, contrary to the positive control group treated with Daboia russelii venom. Nephrotoxicity may result from higher venom doses in the context of coagulopathy, as a complication provoked by venom hematoxicity.

Increased Expression of a Reporter Plasmid by Crotalid Venom Fraction

Increased Expression of a Reporter Plasmid by Crotalid Venom Fraction

Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α2‐adrenoceptors

BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs. EXPERIMENTAL APPROACH In binding experiments with (3) H-rauwolscine, we studied the interactions of green mamba venom fractions with α(2) -adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α(2)-adrenoceptors expressed in mammalian cells. KEY RESULTS ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of (3) H-rauwolscine binding to the three α(2)-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α(2A) -adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA(2) values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively. CONCLUSIONS AND IMPLICATIONS ρ-Da1b is the first toxin identified to specifically interact with α(2)-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α(2)-adrenoceptor subtypes.

Electrophysiological characterization of a novel small peptide from the venom of Conus californicus that targets voltage-gated neuronal Ca2+ channels

Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular ω-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10–15 min produced a significant decrease (54% ± 3%) of the Ca2+ current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca2+ current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca2+ channels.

L-Amino acid oxidase activity present in fractions of Bothrops jararaca venom is responsible for the induction of programmed cell death in Trypanosoma cruzi

Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here we fractionated the venom and observed that the anti- T. cruzi activity was associated with fractions that present l-amino acid oxidase ( l-AAO) activity. l-AAO produces H 2O 2, which is highly toxic. The addition of catalase to the medium, a H 2O 2 scavenger, reverted the killing capacity of venom fractions. The anti- T. cruzi activity was also abolished when parasites were cultured in a medium without hydrophobic amino acids that are essential for l-AAO activity. These results were confirmed with a commercial purified l-AAO. Treatment for 24 h with fractions that present l-AAO activity induced parasites cytoplasmic retraction, mitochondrial swelling and DNA fragmentation, all morphological characteristics of programmed cell death. Similar changes were also observed when parasites were treated with H 2O 2. These results indicate that H 2O 2, the product of l-AAO reaction, induces programmed cell death explaining the anti- T. cruzi activity of B. jararaca venom.

Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom

In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14–31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1–F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of Aα and Bβ chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of Aα and Bβ chains at 5 min and of γ chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm 2/μg, respectively using 25 μg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD 50 of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502–568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126–294 mUA/min/mg). Fibrinolytic activity evaluated with 10 μg protein, showed that B. isabelae venom contained higher specific activity (50 mm 2/μg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.

Selective toxin–lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin–membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3 μg/mL and 43.1 μg/mL against sheep and 13.5 μg/mL and 8.8 μg/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore ® technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4 μg/mL to 35.4 μg/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.

Divergent M- and O-superfamily peptides from venom of fish-hunting Conus parius

Six novel peptides from the piscivorous cone snail, Conus parius were purified by reverse-phase HPLC fractionation of crude venom. With the use of matrix-assisted laser desorption ionization mass spectrometry and standard Edman sequencing methods, the peptides were characterized. Two peptides were identified as members of the m-2 and m-4 branches of the M-superfamily and were designated as pr3a and pr3b, while four peptides were identified as members of the O-superfamily and were designated as pr6a, pr6b, pr6c and pr6d. Peptide pr3a differs from the majority of the M-superfamily peptides in the presence of two prolines, which are not modified to 4- trans-hydroxyproline. In peptide pr3b, five amino acids out of the 16 non-cysteine residues are identical with those of μ-GIIIA and μ-PIIIA, suggesting that pr3b may be a divergent μ-conotoxin. Peptide pr6a is notable because of its extreme hydrophobicity. Peptide pr6c has three prolines that are unhydroxylated. Peptides pr6b and pr6d differ from the previously characterized O-superfamily peptides in the presence of an extended N-terminus consisting of six amino acids. Peptides pr3a, pr3b, pr6a and pr6b were demonstrated to be biologically active when injected intraperitoneally in fish. The identification and characterization of these peptides in venom of a fish-hunting species establish the divergence of gene products and their patterns of post-translational modification within superfamilies in a single Conus species.

Isolation and characterization of a myotoxin from the venom of Macrovipera lebetina transmediterranea

The Macrovipera lebetina venom consists of a complex mixture of proteins belonging to a few main families according to their enzymatic and pharmacological activity. Given the serious pathophysiological effects caused by M. lebetina bites mainly induced by muscle degeneration, we decided to investigate the myotoxic activity of some venom fractions. In the present study we describe the purification and characterization of a 22.600 kDa protein, named in the following Mlp4.2, that shares myotoxic but not haemorrhagic activity in vivo. Herein we report that Mlp4.2 is a metalloproteinase belonging to the PI-SVMPS family able, in vitro, to proteolyse extracellular matrix proteins as laminin and fibronectin. Histological observations of mouse anterior tibialis Mlp4.2-treated muscle, demonstrate that this protein induces a massive degeneration of myofibers but not haemorrhage. The immunofluorescence analysis of protein-treated anterior tibialis, demonstrates that Mlp4.2 is able to disarray the laminin network surrounding muscle fibers. Finally Mlp4.2 did not show any direct cytolytic activity towards the myogenic cell line C2C12 in culture. The data reported herein suggest that the myotoxicity of Mlp4.2 is primarily linked to the disruption of the muscle fibers interaction with extracellular matrix proteins.

Venom components from Citharischius crawshayi spider (Family Theraphosidae): exploring transcriptome, venomics, and function.

Despite strong efforts, knowledge about the composition of the venom of many spider species remains very limited. This work is the first report of transcriptome and venom analysis of the African spider Citharischius crawshayi. We used combined protocols of transcriptomics, venomics, and biological assays to characterize the venom and genes expressed in venom glands. A cDNA library of the venom glands was constructed and used to generate expressed sequence tags (ESTs). Sequence comparisons from 236 ESTs revealed interesting and unique sequences, corresponding to toxin-like and other components. Mass spectrometrical analysis of venom fractions showed more than 600 molecular masses, some of which showed toxic activity on crickets and modulated sodium currents in DmNa(v)1 and Na(v)1.6 channels as expressed in Xenopus oocytes. Taken together, our results may contribute to a better understanding of the cellular processes involved in the transcriptome and help us to discover new components from spider venom glands with therapeutic potential.

Nerve Growth Factor Inhibits Metalloproteinase-Disintegrins and Blocks Ectodomain Shedding of Platelet Glycoprotein VI

Nerve growth factor (NGF) plays an important role in regulating mammalian neuronal/embryonic development, angiogenesis, and other physiological processes and has recently been investigated as a potential treatment for the neurodegenerative disorder, Alzheimer disease. In this study, we provide evidence that human NGF may also function as a metalloproteinase inhibitor, based on studies of NGF from snake venom. Originally, our aim was to isolate snake venom metalloproteinases targeting platelet receptors and/or ligands relevant to hemostasis and thrombosis, using Ni(2+)-agarose as a purification step based on the conserved metal ion-coordination motif in venom metalloproteinases. However, subsequent analysis of cobra (Naja kaouthia) venom led to the unexpected discovery that cobra venom NGF bound to Ni(2+)-agarose, eluting at approximately 15 mm imidazole, enabling a one-step purification. The identity of the purified protein was confirmed by mass spectrometry and N-terminal sequence analysis. Partial co-purification of NGF within metalloproteinase-enriched venom fractions led us to test whether NGF affected metalloproteinase activity. Venom NGF potently inhibited metalloproteinases isolated from the same or different venom and specifically bound to purified Nk metalloproteinase immobilized on agarose beads. Human NGF also interacted with human metalloproteinases because it blocked metalloproteinase-mediated shedding of the platelet collagen receptor, glycoprotein (GP)VI, and associated with recombinant ADAM10 by surface plasmon resonance. Together, these results suggest that NGF can function as a metalloproteinase inhibitor.

Effects of heating on the immunogenicity and biological toxicity of Deinagkistrodon acutus venom and its fractions

To improve toxoid preparation, the effects of selective heat denaturation were assessed on Deinagkistrodon acutus venom. The venom and its fractions (peak 1 and peak 2 separated by gel filtration chromatography) were heated to various temperatures (45–70 °C) for 30 min, after which protein concentration, immunoreactivity, lethality, myotoxicity and hemorrhagic and membrane lysis activities of the samples were determined. In addition, the synergistic effects of the venom fractions were evaluated by separate or simultaneous intramuscular injection in mice. The results showed that the peak 1 fraction consisted primarily of proteins in the range of 18 to 105 kDa, while the peak 2 fraction consisted primarily of proteins smaller than 21 kDa. The hemorrhagic activity, immunoreactivity, and protein concentration of heated samples were gradually reduced as the temperature increased from 25 °C to 70 °C. Bioactivities significantly decreased but immunoreactivity was retained when the crude venom, peak 1 fraction, or peak 2 fraction were heated to the critical temperatures of 60 °C, 55 °C, or 60 °C, respectively. Synergistic effects of two kinds of heated fractions were observed in toxicity and antibody production after the peak 1 and peak 2 injected simultaneously or respectively. The results suggest that venom fractions heated and injected separately could significantly reduce their toxicity and enhance the neutralization of antiserum induced by them.