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A chicken-derived antivenom (ChDAv) towards taipan snake (Oxyuranus scutellatus) venom was produced by purifying anti-taipan IgY from egg yolks of hens immunized with taipan venom. The productivity, antivenomic profile, neutralization ability, pharmacokinetic properties and immunogenicity of the ChDAv were compared with those of an antivenom produced in horses (EDAv). We found that 382 eggs are required to produce the mass of anti-taipan antibodies contained in one liter of equine hyperimmune plasma, and that 63 chickens would be needed to generate the amount of anti-taipan antibodies annually produced by one horse. It was estimated that, in Costa Rica, the production of antitaipan antibodies could be 40% cheaper if chickens were used as immunoglobulin source, instead of horses. During antivenomic assessment, ChDAv showed lower ability to immunocapture the a subunit of taipoxin, the most important neurotoxin in the venom. ChDAv showed a lower ability to neutralize the coagulant and lethal activities of taipan venom. ChDAv was more immunogenic in rabbits than EDAv, probably due to the fact that chickens are phylogenetically more distant to rabbits than horses. This finding may explain why clearance from rabbit bloodstream was faster for chicken-IgY than for equineIgG in a pharmacokinetic study. In conclusion, the production of anti-taipan antivenom was less effective when chicken egg yolks were used as source of immunoglobulins instead of horses. Universidad de Costa Rica/[741-B3-017]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Crotoxin and taipoxin are both neurotoxic phospholipases A 2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A 2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A 2 . Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A 2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca 2+ -free condition, taipoxin or crotoxin inhibits with ic 50 of 20–1000 nM the Na + -dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected. Furthermore, the high-affinity site for [ 3 H]desipramine binding, known to be the norepinephrine transporter, is inhibited with an ic 50 of 14nM by taipoxin independent of phospholipase activity. These results are strong indications that certain synaptosomal biogenic amine transporters are part of the binding proteins for taipoxin and crotoxin. Chemical modification at Tyr-21 of the phospholipase subunit of crotoxin greatly reduces the neurotoxicity and the binding affinity with little effect on the enzymatic activity of the toxin. Hence, Tyr-21 may be an important residue for the binding of crotoxin and perhaps other phospholipase A 2 neurotoxins. Even stronger evidence comes from the finding that replacement of the corresponding Phe residue of bovine pancreatic phospholipase A 2 with Tyr by site-directed mutagenesis enables it to compete with an ic 50 of 1 μM for the binding of [ 125 I]crotoxin, contrasting sharply with the complete lack of such ability with the wild type even at 50 μM. In addition, rat phospholipase A 2 , which has a corresponding Tyr residue, can inhibit the binding of [ 125 I]crotoxin.

Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission. There was no obvious relationship between chain structure and potency, but there was an indication of a relationship between chain structure and binding. The binding of notexin was substantially reversible, the binding of crotoxin was slightly reversible, and the binding of beta-bungarotoxin, taipoxin and textilotoxin was poorly reversible. Experiments with neutralizing antibodies indicated that phospholipase A2 neurotoxins became associated with binding sites on or near the cell surface. This binding did not produce neuromuscular blockade. When exposed to physiological temperatures and nerve stimulation, bound toxin disappeared from accessibility to neutralizing antibody. This finding suggests that there was some form of molecular rearrangement. The two most likely possibilities are: (1) there was a change in the conformation of the toxin molecule, or (2) there was a change in the relationship between the toxin and the membrane. The molecular rearrangement step did not produce neuromuscular blockade. At a later time there was onset of paralysis; the amount of time necessary for onset of blockade was a function of toxin concentration. Phospholipase A2 neurotoxins were not antagonized by drugs that inhibit receptor-mediated endocytosis. In addition, phospholipase A2 neurotoxins did not display the pH-induced conformational changes that are typical of other endocytosed proteins, such as clostridial neurotoxins. However, phospholipase A2 neurotoxins were antagonized by strontium, and this antagonism was expressed against toxins that were free in solution and toxins that were bound to the cell surface. Limited antagonism was expressed after toxins had undergone molecular rearrangement, and no antagonism was expressed after toxin-induced neuromuscular blockade. The cumulative data suggest that phospholipase A2 neurotoxins are not internalized to produce their poisoning effects. These toxins appear to act on the plasma membrane, and this is the site at which they initiate the events that culminate in neuromuscular blockade.

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes. Toxicon 29, 973–988 1991.—Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus , was iodinated and used to demonstrate high affinity, specific binding to guineapig ( Cavia porcellus ) brain synaptosomes and synaptosomal membrane fragments. 125 I-crotoxin binding to the membrane fragments displays two binding plateaus, ( K d1 = 4 nM and K d2 = 87 nM, B max1 = 2 and B max2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau ( K d = 2 nM and B max = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125 I-crotoxin binding to synaptosomal membrane fragments gave a K d -value of 3 nM. The K d value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, β-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A 2 , dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125 I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A 2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.

R. D. Crosland . Effect of drugs on the lethality in mice of the venoms and neurotoxins from sundry snakes. Toxicon 29, 613–631, 1991.—I investigated the efficacy of 10 drugs with respect to reducing the lethality in mice of the following venoms and their respective neurotoxins: Bungarus caeruleus venom; Bungarus multicinctus venom, α-bungarotoxin, β-bungarotoxin; Crotalus durissus terrificus venom, crotoxin; Notechis scutatus scutatus venom; Oxyuranus scutellatus venom, taipoxin. The drugs diltiazem, nicergoline, primaquine, verapamil and vesamicol protected mice from the lethality of B. caeruleus venom, B. multicinctus venom, and/or β-bungarotoxin. Dexamethasone provided protection from B. multicinctus venom, β-bungarotoxin, crotoxin, O. scutellatus venom and taipoxin. Protective activity resided in amphiphilic drugs and correlated with the charge on the drug at physiological pH. Protection from lethality was maximal when the drugs were administered immediately after injection of the venom or toxin. Nifedipine, piracetam and reserpine provided no protection from any of the venoms or toxins tested.

Taipoxin from the Australian taipan Oxyuranus s. scutellatus , was detoxified by reaction with p -bromophenacyl bromide and used for preparing antibodies in rabbits. Of its three subunits only the basic α-chain and the acidic γ-chain evoked antibody production, which may suggest that the neutral β-subunit is hidden in the complex. The antibodies did not cross-react with notexin from Notechis s. scutatus nor did taipoxin cross-react with antivenom against Bungarus multicinctus , in accordance with previous findings. Immunochemical studies show a strong interaction between α and γ, whereas α-β- and β-γ-interactions are weak. In animal experiments no protection was observed when antibodies (in excess) were injected i.v. 5 min after taipoxin. This confirms the irreversibility of the attack, perhaps enzymatic (phospholipase), on vital structures in the nerve terminal membranes and emphasizes the urgency of speedy antisera injections in cases of snake-bite in Australia. The modification with p -bromophenacyl bromide ought to be generally useful in the production of high-titre antibodies against snake venoms in a short time and with no ill effects in the antisera-producing animals.

Polyclonal rabbit antisera were raised against ten snake phospholipase A 2 neurotoxins and one snake phospholipase A 2 cytotoxin. Immunological cross-reactivities between these toxins, two other snake phospholipase A 2 enzymes and pancreatic phospholipase A 2 were studied using ELISA technology. All snake phospholipase A 2 neurotoxins fell into two main antigenic classes. One antigenic class was composed of all the elapid toxins tested (textilotoxin, taipoxin, notexin, pseudexin and β-bungarotoxin), the cytotoxic phospholipase A 2 from Naja naja atra and pancreatic phospholipase A 2 . β-Bungarotoxin seemed to be in an immunological subclass of its own compared to the rest of the elapid toxins. The second antigenic class was comprised of crotalid and viperid phospholipase A 2 neurotoxins (crotoxin, concolor toxin, Mojave toxin, vegrandis toxin, ammodytoxin and caudoxin). Our data indicated that the viperid toxins, caudoxin and ammodytoxin, were an immunological subclass apart from the crotalid toxins.

The presynaptic effects of beta-bungarotoxin, crotoxin and taipoxin were studied in the mouse phrenic nerve-diaphragm preparation (27 degrees C). The phospholipase A2 activity, assayed by pH-stat titration, was reduced to 4-10% at 27 degrees C compared with that at 37 degrees C. The late neuromuscular blocking activity was also reduced by more than three fold for all toxins. In contrast, the early biphasic response to the toxins, i.e. immediate depression followed by facilitation, was not delayed. The evoked quantal release of acetylcholine was enhanced by all toxins at low Ca2+-concentrations during the phase of facilitation, without an increase of the maximal release. At the late phase of treatment with beta-bungarotoxin and taipoxin, the curve relating the quantal contents of endplate potentials with Ca2+-concentration was shifted parallel to the right at low Ca2+, but marked depression of the maximal release occurred at high Ca2+. When diaminopyridine was added at the time of the late phase block by beta-bungarotoxin, the quantal release could still be enhanced at low Ca2+-concentrations, even beyond control; however, the maximal release was not simultaneously restored. It is concluded that the late phase block, but not the early biphasic response, is due to an enzymatic action and the release mechanism is abolished when the hydrolysis of membrane phospholipids proceeds to a certain critical level.

R. D. Crosland . Effect of chlorpromazine and quinacrine on the lethality in mice of the venoms and neurotoxins from several snakes. Toxicon 27, 655–663, 1989.—Antivenoms are the currently available agents for the treatment of intoxication by snake venoms. Therapeutic approaches using more generally available drugs could improve treatment of envenomation by reducing the cost of the therapeutic agent, eliminating hypersensitivity reactions to antivenoms, and reducing storage costs. I investigated the efficacies of chlorpromazine and quinacrine with respect to reducing the lethality in mice of Bungarus caeruleus venom, Bungarus multicinctus venom and its neurotoxic components α- and β-bungarotoxin, Crotalus durissus terrificus venom and its neurotoxic component crotoxin, and Oxyuranus scutellatus venom and its neurotoxic component taipoxin. Venom or toxin was administered i.p., followed immediately by an i.p. injection of chlorpromazine or quinacrine. The effect of drug on the lethality of the venom was recorded 24 hr later. Chlorpromazine and quinacrine were effective antagonists of the lethality of B. caeruleus venom, B. multicinctus venom, and β-bungarotoxin without themselves being overtly toxic. Chlorpromazine (1 mg/kg) increased the ld 50 of B. caeruleus venom, B. multicinctus venom, and β-bungarotoxin by 8.6-, 2.6- and 3.7-fold, respectively. In the range of 1 to 5 mg/kg, quinacrine increased the ld 50 of B. caeruleus venom, B. multicinctus venom and β-bungarotoxin by 5.7-, 11- and 8.6-fold, respectively. Neither drug had any effect on the other venoms or toxins. Chlorpromazine and quinacrine were also injected at different times both before and after the injection of venom or toxin. Protection from lethality was maximal for both drugs when they were administered immediately after injection of venom or toxin.

The venom of the Australian tiger snake Notechis scutatus scutatus has been fractionated by ion-exchange chromatography on Bio-Rex 70 in an ammonium acetate gradient. The main neurotoxic component, the notexin, which constituted 6 per cent of the crude venom, was thereby obtained in a high degree of purity (⩾97 per cent) in one purification step. It is a basic protein of 119 amino acid residues in a single peptide chain cross-linked by seven disulfide bridges. The formula weight is 13,574. The LD 100 is 25 μg/kg mouse. The toxin causes dyspnea and paralysis of the hind legs and in the final stages before death, the mice lie immobilized. The death follows two to three days after the administration of a lethal dose. The notexin is a presynaptic toxin which inhibits the release of acetylcholine from motor nerves. The tiger snake venom also contained four other highly active toxins, three of which were submitted to a preliminary study. The first of these toxins has about 60 amino acid residues and an LD 100 of 100 μg/kg mouse, and the second has about 70 residues and an LD 100 of 150 μg. Both toxins have probably a postsynaptic mode of action, as they appear to have all characteristics in common with the postsynaptic cobra ( Naja naja ) neurotoxins. The third toxin, which contains about 120 residues and has an LD 100 of 600 μg, causes respiratory distress characteristic for a postsynaptic neurotoxin. Sublethal doses can give rise to difficulty in breathing lasting for as long as 48 hr. It resembles the dimers formed during the freeze-drying of cobra neurotoxins. These dimers also cause dyspnea lasting for a long period, whereas the corresponding time for the monomeric cobra toxins is maximally 6 hr.