Abraham I. Louw

The synergism of cardiotoxin and phospholipase A2 in hemolysis

The synergistic effect of exogenous cobra phospholipase A2 on the hemolysis brate of guinea pig erythrocytes by highly purified snake venom cardiotoxins was investigated. In the presence of phospholipase A2 the reaction was not only faster and had a lower activation energy but followed a sigmoidal instead of a linear time course. Similar results were obtained using porcine pancreatic phospholipase A2. Significantly, addition of even a trace of cobra phospholipase A2 (approx. 0.1%, w/w) was sufficient to bring about the full synergistic effect, emphasizing the stringent purity requirements for any meaningful investigation of cardiotoxins own action. The possibility that the action of cardiotoxin on its own may involve the stimulation of an endogenous phospholipase is discussed in the light of the results obtained with exogenous cobra enzyme.

Kinetics of erythrocyte lysis by snake venom cardiotoxins.

Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-automatic method based on light-scattering changes of erythrocyte suspensions at 700 nm which are directly related to hemoglobin release. Small amounts of phospholipase-free cardiotoxin (<100 μg) could be conveniently and rapidly assayed with the high reproducibility in a recording spectrophotometer, and reliable kinetic data were accumulated. Cardiotoxins from two different genera (Hemachatus haemachates and Naja mossambica mossambica) displayed virtually identical hemolytic properties. Hemolysis increased linearly with time, in contrast with a sigmoidal pattern when phospholipase was present as an impurity. Low concentrations of Ca2+ (<1 mM) stimulated cardiotoxin action. A limiting plateau rate of hemolysis reached during concentration dependence experiments in which the level of either cardiotoxin or of erythrocytes was varied, suggested that the interaction of cardiotoxin with erythrocyte membranes is a saturation phenomenon only at a high ratio of cardiotoxin: erythrocytes. No hemolysis was observed with an homologous neurotoxin of S-methylated cardiotoxin, providing evidence for specificity. The linear Arrhenius plots obtained for the temperature dependence of cardiotoxin-induced hemolysis strengthened the conclusion that its action involves more than a detergent-like effect on membrane phospholipids.

Snake venom toxins The amino acid sequences of three cytotoxin homologues from Naja mossambica mossambica venom

Abstract The amino acid sequences of three polypeptides, designated V II 1, V II 2 and V II 3 from Naja mossambica mossambica venom have been determined and differ in altogether five amino acid positions. Except for a single aspartyl residue which is amidated in V II 1 the primary structure of the latter toxin corresponds to that of Toxin γ isolated from the venom of Naja mossambica pallida . When compared to other cytotoxins from the genera Naja and Haemachatus haemachates , V II 1, V II 2 and V II 3 exhibited a high degree of homology. Homology to a lesser degree was also found when the cytotoxins as a group were compared with Neurotoxin α from Naja mossambica pallida .

Snake venom toxins The purification and properties of five non-neurotoxic polypeptides from Naja mossambica mossambica venom

Five non-neurotoxic polypeptides, designated VII1–VII4 and V 5, have been isolated from the venom of Naja mossambica mossambica by column chromatographic techniques. Whole venom was fractionated on Sephadex G-50 and the major fraction, thus obtained, was re-chromatographed on CM-cellulose and Amberlite CG-50 to yield the polypeptides in pure form. They consist of 60 amino acid residues each and have molecular weights of 6820, 6792, 6888, 6712 and 6991, respectively. Immunochemically, polypeptides VII1–VII4 could be identified as cardiotoxins. The position with respect to Polypeptide V 5 was unclear and did not allow a definite assignment. This is the first time that the isolation of more than two cardiotoxins from the venom of the same specie has been described. Polypeptide VII1 was found to have the same amino acid composition as toxin γ, a cardiotoxin that has been isolated from the venom of Naja mossambica pallida.

The conformation of cardiotoxins and neurotoxins from snake venoms

Abstract The conformations of a number of snake venom cardiotoxins and short (60–62 residues) neurotoxins were compared with the aid of circular dichroism spectra, sequences and theoretical predictions. The near-ultraviolet CD spectra of cardiotoxins exhibited positive Cotton effects arising mainly from the side chain of an invariant tyrosine located at position 23 of their sequences. By contrast, neurotoxins without exception had negative near-ultraviolet CD extrema, even though they contain a tyrosine in an identical position. The reversal in CD sign was attributed to the unique presence of tryptophan-28 known to be involved in neurotoxicity and which presumably possesses the necessary negative ellipticity to dominate neurotoxin near-ultraviolet spectra. A prominent positive Cotton effect invariably observed near 230 nm in neurotoxin spectra was likewise assigned to this tryptophan. Despite these characteristic differences, the deep-ultraviolet CD spectra concerned with the conformation of the polypeptide backbone of the toxins were quite similar. All had a single negative extremum near 215 nm, which is indicative of β-conformation and the absence of helix. The comparative CD results and sequence-based theoretical predictions of structure indicated that the diverse biological effects of cardiotoxins and neurotoxins are not due to gross main chain conformational differences. Changes in local side chain structure within a larger common three-dimensional framework are postulated to be sufficient to confer different binding specificities on cardiotoxins and neurotoxins, resulting in their action on different target membranes.

Snake venom toxins the complete amino acid sequence of cytotoxin VII4 from the venom of Naja mossambica mossambica

Summary The primary structure of cytotoxin VII4, consisting of 60 amino acid residues cross-linked by four disulphide bridges, was determined. Cytotoxin VII4 differs in altogether 17 residue positions from the other cytotoxins of the same snake. Comparing the amino acid sequence of cytotoxin VII4 from Naja mossambica mossambica venom to the known sequences of cytotoxins from the genera Naja and Haemachatus , 33 residues, including 8 half cystines, are identical. The amino acid sequence of cytotoxins as a group has 11 residue positions in common with the amino acid sequence of neurotoxins and based on this observation, 8 residues in the primary structure of cytotoxins could be implicated as probably being functionally important.

The importance of protein structure and conformation in the preparation of phospholipase-free cardiotoxin from snake venom☆

Hydrophobic interactions of cobra venom phospholipase A2 (Mr 13 400) in saline with Sephadex gels and its stability towards denaturation in 6 M guanidine-hydrochloride precluded the use of these solvents to remove traces (approx. 0.2%, w/w) of phospholipase A2 from cardiotoxin (Mr 6800) by gel chromatography. Phospholipase-free (less than 0.001%, w/w) cardiotoxin could, however, be obtained by gel chromatography in 8 M urea or 80 mM phenylalanine. Stokes radius and circular dichroism measurements showed that the hydrophobic retardation of phospholipase A2 was abolished but that the hydrodynamic size and conformation of neither protein was affected, thereby facilitating separation.

Correlation between the surface hydrophobicities and elution orders of elapid neurotoxins and cardiotoxins on hydrophobic-interaction high-performance liquid chromatography.

Hydrophilic and hydrophobic regions were predicted for Elapid neuro- and cardiotoxins. The contribution of these regions to the retention times of neuro- and cardiotoxins on hydrophobic-interaction HPLC was assessed from the known surface accessibilities of amino acid side-chains within these regions. Differences in retention times between neuro- and cardiotoxins on hydrophobic-interaction HPLC could be attributed to differences in hydrophobicity of regions 6-12 and 22-26 between these two types of toxins. Smaller differences in retention times between cardiotoxins were due to the variable hydrophobicities of regions 1-4 and 26-36.