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New World elapids are coral snakes that belong to the genus Micrurus, and for which the venom biochemistry is mostly unknown. Analysis has been difficult because the coral snakes produce small quantities of venom. Clinical observations following bites show mainly neurotoxic effects. Experimentally, cardiotoxic, haemolytic and myotoxic activities are also reported. An experimental approach, using reverse-phase high-performance liquid chromatography and specific assays for alpha-neurotoxin and phospholipase A2 activities, was conducted on milligram quantities of venoms from three Micrurus species from Costa Rica; M. nigrocinctus nigrocinctus, M. alleni yatesi and M. multifasciatus. Neurotoxicity was determined by competition binding experiments with the Torpedo marmorata acetylcholine receptor. Phospholipase A2 activity was measured by fluorimetry using a pyrene lipid substrate. In this way, we purified and characterized seven alpha-neurotoxins, five phospholipases A2 and four toxin homologs. The amino acid sequence of the major alpha-neurotoxin from M. nigrocinctus nigrocinctus venom was fully determined and compared to Old Word representatives. Distance matrix data were generated to set up phylogeny relationships among elapid short-chain alpha-neurotoxins, which proved to be in accordance with the taxonomic classification and geographical distribution of snake species.

Danger from snake bites, especially those of Elapidae, pose a public health problem in a large number of tropical and sub-tropical countries. Since the advent of serotherapy, the morality rate has decreased, but suitable sera are not always available, explaining the usefulness of developing symptomatic treatments. The present study is a test of the preventative and curative efficacy of anticholinesterases in the treatment of Naja haje haje venom envenomation. It is clearly shown that the early use of these products leads to a considerable increase in the LD50 in mice having undergone experimental envenomation.

The proton NMR spectra at 300 MHz of neurotoxin III from venom of Naja mossambica mossambica are reported. By the use of double resonance techniques, pH dependence chemical shifts, isotope labeling technique, and comparison with homologous neurotoxins all proton signals in the aromatic and methyl regions as well as epsilon-CH2 proton signals of some lysine residues have been assigned to individual amino acid residues and their spatial microenvironment has been determined. The results deduced on the solution structure of neurotoxin III are in complete agreement with the crystal structure of sea snake erabutoxins as well as with the previously established backbone folding and inter-residue interactions for the Naja naja oxiana short-chain neurotoxin in solution. In addition evidence has been obtained (a) that the conformation of the beta turn in the 31-34 segment depends on the ionization state of Asp-31 and His-32 side chain groups and (b) that an intricate electrostatic interaction exists in a system of ionogenic groups of the invariant Lys-27, Lys-47, Asp-31, Arg-33, Glu-38 and His-32 residues. These aspects of dynamic conformation are related to an interaction mechanism of a neurotoxin molecule and a nicotinic acetylcholine receptor.

Cardiotoxins are small basic proteins (7 000 daltons) that are found in the venoms of Elapidae snakes. Although they are structurally close to alpha-neurotoxins present in the same secretions, their activity is related to their ability to interact with every cell membrane inducing, near micromolar concentration, the modification of its biological properties and/or physical structure. The mode of action of cardiotoxins, on a molecular level, is still under investigation. However, lipid-protein interactions are more and more involved in their binding to membrane and in their activities. Using new experimental data a better definition of phospholipid-cardiotoxin interaction is arrived at and a tentative molecular explanation of the pharmacological activities of these proteins is presented and discussed.

We have compared the effects of highly purified preparations of cardiotoxins and phospholipases A2 from Naja mossambica mossambica venom on rat brain [Na+,K+]-ATPase activity. The results were the following: (i) micromolar concentrations of cardiotoxin preparations were required to inhibit [Na+,K+]-ATPase activity to the extent achieved by picomolar concentrations of phospholipases A2; i.e., the inhibitory effect of cardiotoxins appeared to be related to the contamination of the preparations by trace amounts of phospholipase A2; (ii) comparing phospholipases A2 from varied origins, a correlation was observed between [Na+,K+]-ATPase inhibition, isoelectric point, and toxicity for mice; (iii) when rat brain membranes were used, incubation for extended times with the most basic N. mossambica mossambica phospholipase A2 resulted in a biphasic [Na+,K+]-ATPase inhibition, suggesting that two distinct [Na+,K+]-ATPases were affected differentially. In contrast, incubation of rat brain membranes with either porcine pancreatic phospholipase A2, notexin, or beta-bungarotoxin and also incubation of erythrocyte membranes with the most basic N. mossambica mossambica phospholipase A2 produced monophasic [Na+,K+]-ATPase inhibitions. We discuss a possible specific action of toxic, basic phospholipase A2 on one of the [Na+,K+]-ATPase isoforms of excitable membranes.

Cardiotoxins are small basic proteins which cause heart failure when they are injected in vivo. In order to better understand their molecular mode of action, short peptides designed on the model of the first loop of the molecule of cardiotoxin IV from Naja mossambica mossambica venom have been synthetized by the solid-phase procedure of Merrifield. These peptides express lethality in mouse when they are injected intravenously. Taking into account the respective molecular weights, they are 3.5 to 5% as toxic as the cardiotoxin. Furthermore, the symptomatology they induce is undistinguishable from that induced by cardiotoxins. These results strongly support our previous hypothesis that the first loop of the molecule is the toxic site of cardiotoxins.

The interaction of cardiotoxin II of Naja mossambica mossambica with cardiolipin model membranes was investigated by binding, fluorescence, resonance energy transfer, fluorescence quenching, 31P NMR, freeze-fracture, and small-angle X-ray experiments. An initially electrostatic binding appeared to be accompanied by a deep penetration, most likely into the acyl chain region of the phospholipids, indicating a hydrophobic contribution to the strong interaction (KD congruent to 5 X 10(-8) M). This binding results in a fusion of unilamellar vesicles as indicated by a fluorescence-based fusion assay, freeze-fracture, and X-ray diffraction. In these fused structures freeze-fracture electron microscopy reveals the appearance of particles, which is accompanied by the induction of an isotropic component in 31P NMR. The well-defined particles are interpreted as inverted micelles, and the localization of the cardiotoxin molecule in these structures is discussed.

Surface fluorescence spectroscopy was used to study mixed phospholipid-cardiotoxin monomolecular films. Using polarized incident light we were able to detect the intrinsic fluorescence emission at a wavelength of 339 nm of the single Trp11 in the cardiotoxin molecule. Its fluorescence intensity increased continuously upon film compression up to a maximal value reached at 42 mN x m-1 in surface pressure. A relative polarization index can be used as an indication of the orientation of the indole ring. A transition at around 25 +/- 5 mN x m-1 in the orientation of the Trp11 relative to the plane of the interface was clearly evidenced during film compression, in agreement with our independent data showing a flipping of the whole toxin molecule (Bougis et al., (1981), Biochemistry 20, 4915-4920).

The amino acid sequences of two neurotoxins of the African cobra Naja mossambica mossambica have been determined using almost uniquely phenylisothiocyanate degradation in a liquid protein sequencer programmed alternatively with 'protein' and 'peptide' programs. When compared to known sequences of so-called 'short' neurotoxins belonging to other Elapidae snakes, neurotoxins I and III of Naja mossambica mossambica are very similar to the cobrotoxin, a neurotoxin isolated from the formosan cobra Naja atra atra.

Using the previously reported sequence-specific 1H-NMR assignments, structural constraints for the cardiotoxin CTXIIb from Naja mossambica mossambica were collected. These include distance constraints from nuclear Overhauser enhancement measurements both in the laboratory and in the rotating frame, dihedral angle constraints derived from spin-spin coupling constants, and constraints from hydrogen bonds and disulfide bridges. Structure calculations with the distance geometry program DISMAN confirmed the presence of the previously identified antiparallel beta-sheets formed by residues 1-5 and 10-14, and by 20-27, 35-39 and 49-55, and established the nature of the connections between the individual beta-strands. These include a right-handed crossover between the two peripheral strands in the triple-stranded beta-sheet, and a type I tight turn immediately preceding the beta-strand 49-55. The spatial arrangement of the polypeptide backbone in the solution structure of CTXIIb is closely similar to that in the crystal structure of the homologous cardiotoxin VII4 from the same species. In an Appendix the origin of the large pH dependence of two amide proton chemical shifts in CTXIIb is explained.

The vast majority of Elapidae snake venoms, genus Naja, includes three classes of toxic polypeptides: alpha-neurotoxins, phospholipases A2, and cardiotoxins. A new experimental approach using reverse-phase high-performance liquid chromatography in particular has been developed, allowing their respective resolution, identification, and quantitation from milligram quantities of venom. First, definition of optimal chromatographic conditions for Naja mossambica mossambica toxins has been ascertained. Different column packing and solvent systems were compared for their efficiency, with particular attention to the ionic strength of the aqueous solvent. A medium-chain alkyl support (octyl) in conjunction with a volatile ammonium formate (0.15 M, pH 2.70)/acetonitrile solvent system was found to be particularly effective. All the components known until now from this venom could be resolved in a single step, and the elution order was alpha-neurotoxins, phospholipases A2, and cardiotoxins with a total recovery of absorbance and toxicity. Then, with these suitable conditions, we describe a new major cardiotoxin molecule in this venom by hydrophobic and not ionic-charge discrimination. Second, specific assays were designed to detect alpha-neurotoxin and phospholipase A2 activities in chromatographic fractions: alpha-neurotoxin activity was determined by competition for the binding of a radiolabeled alpha-neurotoxin to the acetylcholine receptor of the ray electric organ, and phospholipase A2 activity was defined by the enzymatic activity of these toxins with a fluorescent phospholipid as substrate. Finally, the applicability of these new methods to study other Naja snake venoms was demonstrated.

The monomolecular film technique was used in order to study the specific interactions of 4 cardiotoxins from Elapidae snake venom Naja mossambica mosambica with different phospholipids. The interaction, at pH 7.5, of cardiotoxin (10(-7) M) with both neutral and negatively charged phospholipids occurs up to a very high critical surface pressure (pi = 45 dynes/cm with the latest). The apparent molecular area of cardiotoxin molecule, during its insertion into a negatively charged phospholipid film, presents only two characteristic values: 1400 A2 for pi less than 20 dynes/cm and 420 A2 for pi greater than 30 dynes/cm, the transition occurring in a very narrow range of surface pressure (25 +/- 5 dynes/cm). Thus, according to the surface pressure, the cardiotoxin may present two different orientations: "flat" or "edgewise" and the transition between both could account for lytic activity of cardiotoxin.

The lethality of elapid snake venoms is due to toxic polypeptides which are alpha-neurotoxins, phospholipases A2 and cardiotoxins. In contrast to alpha-neurotoxins, less is known about the mode of action of phospholipases A2 and cardiotoxins at a cellular and molecular level, although it has been demonstrated that the hemolytic effect of cardiotoxin and its action on muscle can be accelerated in vitro by phospholipase A2. Here we show that when mice are injected i.v. with phospholipase A2, a significant decrease in survival time is observed if a sub-LD50 amount of cardiotoxin is injected simultaneously. Furthermore, the survival time is drastically reduced when phospholipase A2 (2.3 LD50) is injected first, followed 15 min later by doses of cardiotoxin as small as 0.18 LD50. These results strongly suggest a synergy between the two polypeptides.

Four cardiotoxins (CTX I-IV) from Naja mossambica mossambica were compared for their ability to interact with phospholipid vesicles and their capacity to bind erythrocytes. It is concluded that the affinity of the toxins always increases in the order: I approximately equal to II less than III less than IV. The binding is specific for charged lipids even in lipid mixtures. Proteolytic attack of the free and lipid-bound cardiotoxin indicates that at least the first loop Leu1-Thr13 is at the lipid contact. Tryptic and synthetic peptides constitutive of this loop are shown to interact with lipids. Arg5 residue increases the affinity toward the bilayer. The Raman spectra of lipid-bound cardiotoxin indicate a secondary and tertiary structure mainly similar to that of the free toxin. On charged lipids cardiotoxins induce a decrease of the enthalpy and an increase of disorder without change in the transition temperature; at saturating amounts of toxin the transition is abolished. In binary mixtures of phosphatidylcholine and charged lipids the observed effects can be accounted by a phase separation induced by the toxin.

The monomolecular film technique was used to compare the specific interaction of four cardiotoxins from Naja mossambica mossambica with different phospholipids. We were able to demonstrate the interaction of cardiotoxins (10(-7) M) with both neutral and negatively charged phospholipids up to very high surface pressures (45 dyn/cm). In the presence of a phospholipid monolayer, the surface activity of cardiotoxins became much greater than that observed at the air-water interface. Neurotoxins of the same venom do not penetrate a phospholipid film, even at low surface pressure (15 dyn/cm). The apparent molecular area of cardiotoxin III during its insertion into a negatively charged phospholipid film was quantitatively defined. As a function of surface pressure of the membrane around 25 dyn/cm, cardiotoxins may exist in two different configurations, "flat" (1400 A2) or "edgewise" (420 A2). This result could account for the lytic activity of this type of toxin.