Antibodies to sea anemone nematocyst venom—I. Serological demonstration of specific antibodies produced in rabbits in response to nematocyst venom from the sea anemone, Aiptasia pallida

R. I. Grove and D. A. Hessinger. Antibodies to sea anemone nematocyst venom—I. Serological demonstration of specific antibodies produced in rabbits in response to nematocyst venom from the sea anemone, Aiptasia pallida. Toxicon17, 99–108, 1979.—Sea anemone, Aiptasia pallida, nematocyst venom was attenuated by dialysis against dilute formaldehyde and in combination with complete Freund's adjuvant used to immunize laboratory rabbits. Subsequent immunizations (challenges) were administered using active venom with incomplete Freund's adjuvant. The kinetics and strengths of the elicited humoral immune responses were monitored using double immunodiffusion in two dimensions (Ouchterlony technique) and microimmunoelectrophoresis. Subsequent immune challenges were administered only at optimal intervals after the previous immunization as determined by the loss of detectable precipitin reactions on Ouchterlony plates. The antisera of all challenges reacted identically with active and attenuated venoms indicating that the attenuated venom was antigenically equivalent to the active venom in in vitro immunodiffusion reactions and immunogenically equivalent in vivo in its ability to elicit humoral immune responses.     

Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipases A2

Carbamylation of 9 out of 10 lysine residues in the toxic phospholipase A₂ from N. nigricollis venom decreased its lethality at least 8-fold and abolished its direct hemolytic and anticoagulant activities, while the enzymatic activity, as measured on purified substrates, decreased only about 50%. Likewise, carbamylation of 3 out of 5 lysines in the relatively less toxic N. naja atra phospholipase induced detoxification and caused a loss of its blocking activity on the phrenic nerve-diaphragm preparation, while its enzymatic activity on purified substrates was unaltered. Results obtained when 7.4 out of 10 lysines in N. nigricollis phospholipase were carbamylated indicate that basicity is not an absolute requirement for high lethal potency, hemolytic activity or cardiotoxicity.The extent of phospholipid hydrolysis induced in erythrocytes, rabbit plasma, phrenic nerve-diaphragm preparation, brain minces and brain synaptic plasma membranes by incubation with the carbamylated enzymes was in agreement with their enzymatic activities as measured on purified substrates. Levels of phospholipid hydrolysis in heart, lung and kidney of mice given phospholipase intravenously, and in brain synaptic plasma membranes from rats given phospholipase intraventricularly, showed that carbamylated derivatives of N. nigricollis phospholipase A₂ lost their ability to reach and/or hydrolyze substrates in vivo. However, the decrease in in vivo phospholipid hydrolysis did not correlate with the decrease in toxicity since, at comparably low levels of phospholipid hydrolysis, some phospholipases were lethal and others were not. Moreover, when intraventricularly administered, both lethal amounts of the native N. naja atra enzyme and its nonlethal carbamylated derivatives produced equally low hydrolysis of synaptic membrane phospholipids.By means of lysine carbamylation, a dissociation between hydrolytic activity and pharmacological properties of phospholipases A₂ has been achieved. We suggest, therefore, that the toxicity of pure phospholipases is primarily due to a direct effect which does not correlate with levels of phospholipid hydrolysis and that this direct effect is prominent in the relatively toxic phospholipases while it is less manifest in the relatively non-toxic enzymes.     

Isolation and characterization of a toxic phospholipase A2 in the spitting cobra (Naja mossambica mossambica) venom.

N. Martin-Moutot and H. Rochat. Isolation and characterization of a toxic phospholipase A₂ in the spitting cobra (Naja mossambica mossambica) venom. Toxicon17, 127–136, 1979.—A phospholipase A₂ from the spitting cobra (Naja mossambica mossambica) was purified by chromatographic procedures involving gel filtration on Sephadex G-50 and ion exchange on Amberlite IRC-50. The purified enzyme was homogeneous according to physico-chemical criteria as well as chemical purity tests. This protein was characterized by amino acid composition, determination of N- and C- terminal amino acid sequences, isoelectric pH, molecular weight. It exhibits phospholipase activity and toxicity to mice. Positional specificity towards phosphatidylcholine showed that it belongs to the A₂ type. Optimal activity was observed at an alkaline pH and calcium was required for phospholipase activity.     

A comparison of the toxinological characteristics of two Cassiopea and Aurelia species.

A comparison of the toxinological properties of nematocyst venoms from Old and New World Cassiopea and Aurelia species was undertaken. The cnidom of venomous Cassiopea andromeda (Ca) and Aurelia (Aa_RS) from the Red Sea was identical to that of nonvenomous Bahamian Cassiopea xamancha (Cx) and Chesapeake Bay Aurelia aurita (Aa_CB), respectively. A clean nematocyst preparation of Ca and both Aurelias could be obtained but algal particles could not be separated completely from the Cx nematocysts. Further purification of all four nematocyst preparations showed significant differences in the action of their protein. Only the Cassiopea had coexisting dermonecrotic and vasopermeability producing properties and Ca’s hemolytic activity was associated with mouse lethality. The protein, hemolysin and phospholipase gel filtration eluant curves of Ca venom were similar. Venomous Aa_RS actively stung lips and contained more potent mouse lethal, demonecrotic, vasopermeability plus hemolytic factors than Aa_CB. Cross reactivity of convalescent human serum obtained from patients stung by Ca and venomous Cx collected in Central America occurred. This was also observed between sera of bathers stung by Aa_RS and stinging Aurelia which appeared in Florida during the recent El Niño year. IgG was stimulated by several nematocyst proteins since many venom subfractions tested positive at high titers against convalescent sera. T-cell proliferation of mice primed with either Aurelia venom was positive against the homologous preparation with cross reactivity to the heterologous venom. Crude venoms of both Red Sea jellyfish metabolically stimulated cultured human hepatocytes more than their New World counterparts. This data shows that considerable similarities and differences exist in the venoms of these Old and New World Cassiopea and Aurelia medusae with the Eastern species being more potent.     

Degradation of red cell membrane phospholipids by sea anemone nematocyst venom

Sea anemone (Aiptasia pallida) nematocyst venom exhibited phospholipase A activity on red cell membrane phospholipids. Phospholipase A, purified from the venom, converted four of the major membrane phospholipids to their respective lysoderivatives, and to water-soluble phosphorus compounds. The production of these water-soluble derivatives appears to be due to the action of a lysolecithinase intrinsic to red cell membranes. Thus, the extent of membrane phospholipid hydrolysis by venom phospholipase A was determined either by direct measurements of the decrease in phospholipid content, or by adding the increase in lysophospholipids to the increase in water-soluble phosphorus compounds. These findings suggest that the underlying mechanism of nematocyst venom-induced hemolysis is due to the enzymatic action of venom phospholipase A on membrane phospholipids.     

Purification and partial characterization of the phospholipase A2 and co-lytic factor from sea anemone (Aiptasia pallida) nematocyst venom

Functional nematocysts of one specific morphological class, the penetrant microbasic mastigophores, were isolated from the sea anemone, Aiptasia pallida. These nematocysts contain a multicomponent venom composed of several proteins, including those with neurotoxic, hemolytic, and lethal activities. Hemolytic activity is produced by at least three synergistic venom proteins. One of these proteins is identified as a phospholipase A₂ (EC 3.1.1.4) which exists in two isozymic forms, α and β, with molecular weights of 45,000 and 43,000, respectively. The β isozyme has been purified to homogeneity. It is a single-chained glycoprotein with an isoelectric point (pI) of 8.8 and represents 70% of the phospholipase activity of the venom. The activity of the β isozyme is relatively labile and is inactivated by 3.5 M urea or by heating at 45°C. It is most stable at pH 4.0 and loses 50% of its activity at pH values below 3.5 and above 8.0. A second venom protein has also been purified. It is essential for the hemolytic activity of the venom and is termed co-lytic factor (CLF). It is a monomeric glycoprotein having a pI of 4.5. CLF has a molecular weight of approximately 98,000, a sedimentation coefficient of 4.8 S, and is prolate in shape, having a frictional ratio of about 1.6. CLF constitutes about 1.25% of the total venom protein and is assayed by reversing fatty acid inhibition of the venom hemolysis activity.     

Isolation and characterization of the main toxic fraction from the venom of the false horned viper (Pseudocerastes fieldi)

R. Batzri-Izraeli and A. Bdolah. Isolation and characterization of the main toxic fraction from the venom of the false horned viper (Pseudocerastes fieldi). Toxicon20, 867–875, 1982—The venom of Pseudocerastes fieldi was subjected to gel filtration on Sephadex G-75. Most of the protein and lethality of the venom were eluted in a major symmetrical peak (C). The lethality of this peak is confined to a basic protein fraction, Cb (pI > 9.5) separable by DEAE-cellulose chromatography. Two proteins with molecular sizes close to 16,000 daltons were isolated from this fraction by preparative acidic gel electrophoresis in the presence of Triton X-100. One of the proteins (CbII) is lethal to mice ($\text{ld}{}_{50}\text{= 1}\text{mg/kg}$) and shows phospholipase A activity as well as direct hemolytic activity. The other protein (CbI) does not reveal any known biological activity. However, upon recombination of the two a synergistic lethal activity is evident ($\text{the}\text{ld}{}_{50}\text{of the mixture = 0.25 mg/kg}$). It is suggested that CbI may be a specifier which potentiates the toxicity of the phospholipase A at the target site.     

A comparison of the toxicology of the nematocyst venom from sea nettle fishing and mesenteric tentacles

Sea nettle (Chrysaora quinquecirrha) nematocyst venoms from the fishing and mesenteric tentacles were studied. Fishing tentacle nematocyst venom was more potent than mesenteric tentacle nematocyst venom in producing human cutaneous pain, mouse lethality, hemolysis, dermonecrosis, vasopermeability and damage to rat liver mitochondria. Both types of venom had similar enzyme contents except that mesenteric tentacle venom contained no hyaluronidase. The mouse lethal factors of both venoms had a molecular weight probably over 100,000, were not precipitated by 20% (NH₄)₂SO₄ and could be further fractionated by gel electrophoresis and isoelectric focusing.     

Immunosorbent chromatography of sea nettle (Chrysaora quinquecirrha) venom and characterization of toxins

C. S. Cobbs, P. K. Gaur, A. J. Russo, J. E. Warnick, G. J. Calton and J. W. Burnett. Immunosorbent chromatography of sea nettle (Chrysaora quinquecirrha) venom and characterization of toxins. Toxicon21, 385 – 391 1983. — A lethal toxic fraction from nematocysts of the sea nettle (Chrysaora quinquecirrha) fishing tentacle was partially purified by immunochromatography using an immobilized monoclonal antibody column. Elution from the immunosorbent was accomplished under mild conditions which conserved the biological activity of the toxin. The isolated fraction, which contained two purified protein bands with molecular weights of 100,000 and 190,000 daltons on SDS polyacrylamide gels, was both cardiotoxic and neurotoxic and exhibited an intravenous lethal activity (ld₅₀) of 0.37 μg/g in mice.     

Estudio comparativo de los venenos de serpiente Cascabel (Crotalus durissus durissus) de ejemplares adultos y recien nacidos

B. Lomonte, J. A. Gené, J. M. Gutiérrez and L. Cerdas. Estudio comparativo de los venenos de serpiente Cascabel (Crotalus durissus durissus) de ejemplares adultos y recién nacidos. Toxicon21, 379 – 384, 1983. — Venoms from adult and newborn Central American rattlesnakes (Crotalus durissus durissus) were compared for lethal, proteolytic, hemorrhagic, myonecrotic, edematigenous and in vitro hemolytic activities. Electrophoretic and immunoelectrophoretic patterns showed some differences between these venoms. Venom from newborn snakes was devoid of hemorrhagic and edematigenous activities, whereas the venom from adult specimens induced these effects. On the other hand, newborn snake venom showed higher lethality and indirect hemolytic activity, and lower proteolytic activity, than venom from adult specimens. Both types of venoms induced only slight myonecrosis in mice, as judged by histological observation. The ed₅₀ of an antivenom, in terms of absolute weight neutralized per ml of serum, was lower for the newborn specimens venom than for adult's venom, however, for each venom the number of ld₅₀ neutralized was similar.     

Isolation and in vitro partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish Stomolophus meleagris

Jellyfish venom contains various toxins and can cause itching, edema, muscle aches, shortness of breath, blood pressure depression, shock or even death after being stung. Hemolytic protein is one of the most hazardous components in the venom. The present study investigated the hemolytic activity of the nematocyst venom from jellyfish Stomolophus meleagris. Anion exchange chromatography, DEAE Sepharose Fast Flow, and gel filtration chromatography, Superdex200 had been employed to isolate hemolytic proteins from the nematocyst venom of jellyfish S. meleagris. Hemolysis of chicken red blood cells was used to quantify hemolytic potency of crude nematocyst venom and chromatography fractions during the purification process. Native-PAGE profile displayed one protein band in the purified hemolytic protein (SmTX); however, two protein bands with apparent molecular weights of ～45 kDa and 52 kDa were observed in the reducing SDS–PAGE analysis. Approximately 70 μg/mL of SmTX caused 50% hemolysis (HU₅₀) of the erythrocyte suspension. The hemolytic activity of SmTX was shown to be temperature and pH dependent, with the optimum temperature and pH being 37 °C and pH 5.0. The present study is the first report of isolation and partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish S. meleagris. The mechanism of the hemolytic activity of SmTX is not clear and deserves further investigation.     

Synergism between baltergin metalloproteinase and Ba SPII RP4 PLA2 from Bothrops alternatus venom on skeletal muscle (C2C12) cells

Acute muscle damage, myonecrosis, is one of the main characteristics of envenoming by Bothrops genus. In this in vitro study we investigated the role of a metalloproteinase (baltergin) and an acidic phospholipase A₂ (Ba SPII RP4) in the cytotoxicity exhibited by Bothrops alternatus venom. Baltergin metalloproteinase purified from the venom exerted a toxic effect on C2C12 myoblast cells (CC₅₀: 583.34 μg/mL) which involved morphological alterations compatible with apoptosis/anoikis. On the contrary, the most abundant PLA₂ isolated from this venom did not exhibit cytotoxicity at times and doses tested. However, when myoblasts were treated with both enzymes together, synergic activity was demonstrated. Neutralization of the venom with specific antibodies (IgG anti-baltergin and IgG anti-PLA₂) confirmed this synergism.     

Ethoxyformylation and guanidination of snake venom phospholipases A2: Effects on enzymatic activity,lethality and some pharmacological properties

Lysine residues in the basic and relatively toxic N. nigricollis phospholipase A₂ and in the acidic and relatively non-toxic N. n. atra phospholipase A₂ were modified by acylation with ethoxyformic anhydride (in the presence or absence of the substrate dihexanoyl lecithin) or guanidination with O-methylisourea. Ethoxyformylation gave rise to some protein fractions in which enzymatic activity was preserved to a greater degree than intraventricular lethality. Guanidination had little effect on the isoelectric point or catalytic activity of either enzyme or on the lethal potency of the N. n. atra enzyme. However, the intraventricular lethality of the N. nigricollis enzyme was decreased much more than was its intravenous lethality, direct hemolytic potency, anticoagulant activity or cardiotoxic action on rat atrium. These results are compared to those previously obtained when the lysines in these two enzymes were carbamylated with potassium cyanate, a procedure which markedly decreased the isoelectric point of the enzymes. It is concluded that charge alone does not account for differences in toxicity. The data also indicate that there are at least two distinct active sites in both enzymes, one being primarily responsible for enzymatic activity and the other(s) associated with lethal and pharmacological effects of the protein. Modification of lysines affects the latter site(s), while having little or no effect on enzymatic activity.     

The relationship of the possible allergic response to jellyfish envenomation and serum antibody titers

The sera of patients envenomated by the sea nettle (Chrysaora quinquecirrha) or the Portuguese man-o'war (Physalia physalis) were investigated for immune specific and cross-reacting antibodies. Crude or partially purified (SP-Sephadex column chromatography) nematocyst venom was used as antigen in an enzyme-linked immunosorbent assay (ELISA) designed to detect IgG and IgE antibodies. The sera of 66 patients who exhibited strictly cutaneous, extracutaneous or anaphylactoid reactions to envenomation were studied. Most of the subjects developed an IgG antibody and many developed an IgE antibody to the venom of the offending animal. The titer of both immunoglobulins correlated directly with the severity of symptoms. Cross-reacting antibodies to these two jellyfish were apparent in a significant number of patients, but detectable cross-reacting IgE antibodies were rare in patients severely stung by the sea nettle. The titer of specific IgG antibody was higher against the partially purified lethal sea nettle venom than fractions lacking lethal activity. These results may support the hypothesis that some of the visible response to jellyfish envenomation may be allergic in nature and that cross-reactivity to these venoms may be clinically important.     

Central neurotoxicity of cobra neurotoxin,cardiotoxin and phospholipase A2

Central neurotoxicity of cobra neurotoxin (cobrotoxin), cardiotoxin and phospholipase A₂ isolated from Naja naja atra venom was studied in rats and cats by comparing their lethality and death times following central and peripheral applications. On central application, phospholipase A₂ was most toxic, followed by cardiotoxin, with cobrotoxin being least toxic. The time to death was, in general, much longer by central than by peripheral injection. Cardiotoxin, almost immediately after central application, and phospholipase A₂, after a delay, produced recurrent convulsions. Only mild convulsive movements of the head or extremities were observed with cobrotoxin. The respiratory discharge down the phrenic nerve could be recorded even after complete paralysis of respiratory muscles upon central application of cobrotoxin. From these results, it is concluded that phospholipase A₂ and cardiotoxin, rather than the neurotoxin, are responsible for the high toxicity of cobra venom on central application and that the cause of death by the neurotoxin is peripheral in origin even after central application.     

Relationship between catalysis and toxicological properties of three phospholipases A2 from elapid snake venoms

Phospholipases A₂ from three elapid snake venoms were studied in order to determine if differences in toxicity correlate with differences in pattern or level of phospholipid hydrolysis. The comparatively toxic basic phospholipase A₂ isolated from Naja nigricollis venom exhibits a cardiotoxic action following iv administration in mice that is not exhibited by the less toxic neutral phospholipase A₂ from Hemachatus haemachatus venom or the acidic phospholipase A₂ from Naja naja atra venom. This cardiotoxic action correlates with high levels of phosphatidylserine hydrolysis in heart. Levels and patterns of phospholipid hydrolysis in heart, lung and kidney following iv administration suggest that only the N. nigricollis enzyme has the ability to penetrate permeability barriers in the heart. No cardiotoxic effects are seen following intraventricular injection of a lethal dose of the phospholipases A₂. All three phospholipases A₂ (12 μg/ml) abolish the directly and indirectly elicited muscle twitches of the rat phrenic nerve-diaphragm preparation. This block, in normal or altered bathing media, appears to correlate with the level of phospholipid hydrolysis for the N. naja atra enzyme, but not for the N. nigricollis enzyme. These results suggest that N. nigricollis phospholipase A₂ acts by another mechanism in addition to phospholipid hydrolysis.     

Isolation of proteinase inhibitory,toxic and hemolytic polypeptides from a sea anemone, Stoichactis sp.

From a sea anemone, Stoichactis sp., biologically active polypeptides exhibiting toxic, hemolytic and proteinase inhibitory properties have been isolated by gel filtration and ion-exchange chromatography. For the hemolytic polypeptide a mol. wt of 10,000 was determined by gel filtration. It induces complete hemolysis of human erythrocytes at 0·86 μg/ml concentration, is free of phospholipase A activity and has ichthyotoxic activity. The toxic principle (minimum lethal dose ca. 2·3 mg/kg injected s.c. into mice, and 0·5 mg/kg for crabs, i.m. injection) has a mol. wt of about 6000 and was separated from the hemolysin and from the three proteinase inhibitors which inhibit trypsin and chymotrypsin and which are composed of 52–56 amino acid residues.     

Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—II: Pharmacological properties in relationship to enzymatic activity

Despite a remarkable degree of homology in amino acid sequence, the neutral phospholipase A₂ from Hemachatus haemachatus venom is much less toxic than the basic phospholipase A₂ from Naja nigricollis venom, the i.v. ld₅₀ in mice for the two being, respectively, 8.6 and 0.63 mg/kg. Similarly following intraventricular injection into rats, the neutral phospholipase showed convulsant and lethal dose₅₀ values of about 7.5 and 15 μg per rat, respectively, whereas corresponding values for the basic phospholipase were 0.5 and 0.5 μg per rat. Death appears to be due to congestion, hemorrhage and edema in the lungs. Consideration of dosages required and times until onset of action suggests that, dependent upon the route of administration, the effect is either mediated via a central action or is due to a direct effect on the cardiac and/or respiratory system in the periphery. The pattern and extent of phospholipid hydrolysis in various brain regions was similar following intraventricular injection of the two phospholipases so that no relationship between phospholipid hydrolysis and lethal potency could be established. Concentrations of 5 and 10 μmg/ml of the N. nigricollis and H. haemachatus phospholipases, respectively, were required to block electrical activity of the isolated single electroplax. The ultrastructural changes produced by both phospholipases were also similar. Parallel to the somewhat greater potency on the electroplax, N. nigricollis phospholipase produced slightly greater overall hydrolysis in the innervated and non-innervated membranes of the electroplax than did H. haemachatus phospholipase. The results suggest that these two phospholipases do not have a specific junctional effect and that the small difference in potency on the junction cannot be responsible for the large difference in lethality observed in mammalian species.     

Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer

J. M. Gutiérrez, B. Lomonte and L. Cerdas. Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer. Toxicon24, 885 – 894, 1986. — A myotoxin from the venom of the snake Bothrops nummifer was purified to homogeneity by ionexchange chromatography on CM-Sephadex. The toxin is a basic dimer with a subunit molecular weight of 16,000, as estimated by SDS-polyacrylamide gel electrophoresis. The toxin lacks phospholipase A₂ activity when tested on egg yolk lecithin and skeletal muscle homogenates. It induces skeletal muscle damage both in vivo and in vitro. When injected i.m. it promotes a drastic increase in serum creatine kinase levels; the isozyme CK-MM is responsible for this increment. A rapid release of creatine kinase was observed when mouse gastrocnemius muscle was incubated with the toxin, suggesting that it induces the formation of relatively large ‘lesions’ in the plasma membrane of muscle cells. Moreover, analysis of the dose - response data indicated that the myotoxin affects muscle sarcolemma by a ‘one hit’ mechanism. Skeletal muscle cells are affected by the toxin when calcium is eliminated from the medium. The myotoxin has an i.v. ld₅₀ of 3.9 mg/kg body weight in mice, and induces edema when injected in the foot pad. On the other hand, it is not directly hemolytic, anticoagulant, hemorrhagic nor cytotoxic for lymphocytes. The myotoxin shows partial immunologic identity with a myotoxic phospholipase A₂ isolated from Bothrops asper venom. The polyvalent antivenom produced in Costa Rica forms a precipitation arc against B. nummifer myotoxin on immunoelectrophoresis.     

Carbamylation with cyanate of basic phospholipase A2 from the venom of Naja nigricollis (Spitting cobra)

The most basic and toxic phospholipase A₂ purified from Naja nigricollis venom was subjected to lysine modification with cyanate at pH 8.0. The carbamylated derivatives were separated by chromatography on a column of SP-Sephadex C-25 and five fractions were obtained. Amino acid analysis showed that the numbers of Lys-residues modified for fractions Fa to Fe were 10, 9.2, 9.0, 8.2 and 7.4 respectively. The pI values decrease with increasing carbamylation, converting the basic enzyme into an acidic protein. It is noteworthy that even after 9 out of 10 Lys-residues had been modified (Fc) and the pI of the enzyme decreased from 10.6 to 4.4, the enzyme still possessed 75% of its enzymatic activity and 51% of its antigenicity. Although both native and Lys-modified N. nigricollis phospholipase A₂ were perturbed by the presence of Ca²⁺, the difference spectra of Lys-modified Fd differ greatly from that of the native enzyme and become similar to that of the acidic and less toxic phospholipase A₂ from N. naja atra venom. At pH 8.0, the effect of Ca²⁺ on the fluorescence emission intensity of 8-anilinonaphthalenesulfonate-Lys-modified Fd complex was different from that of 8-anilinonaphthalenesulfonate-native N. nigricollis phospholipase A₂ complex and was similar to that of 8-anilinonaphthalenesulfonate-_{N. naja atra} phospholipase A₂ complex. It is clear that the different conformational changes induced by Ca²⁺ might be attributable to the charge properties of the enzyme, which depend on the pH of the solution and the pI of the enzyme. These data are discussed in relationship to the decreased lethal potency and pharmacological effects induced by carbamylation (Condreaet al., 1981b).