Charles Kopeyan

Primary structure of scorpion anti-insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus

The amino acid sequences of insect‐selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S‐carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1 (70 residues) show the shift of one half‐cystine from an external position, which is characteristic of anti‐mammal toxins, to an internal sequence position. Lqq IT2, (61 residues) displays the half‐cystine residue in position 12, common to the sequence of all known antimammal toxins; it induces flaccid paralysis on insects but is non‐toxic for the mouse. Lqq IT2, structurally defines a new type of anti‐insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.

Amino acid sequence of neurotoxin V from the scorpion Leiurus quinquestriatus quinquestriatus.

From the 5 neurotoxins active on mammals found in the venom of Leiurus quinquestriatus quinquestrkztus, toxin V is quantitatively the most important and qualitatively the most potent [l] . It consists of a single polypeptide chain of 64 amino acid residues cross-linked by 4 disulfide bridges. Taking into account preliminary results obtained with the reduced and S-methylated protein (26 residues from the N-terminus) it was classified in the same group of scorpion neurotoxins as neurotoxin II of Androctonus australis Hector [2] . We report here the complete amino acid sequence of neurotoxin V of Leiurus quinquestriatus quinquestriatus which was worked out using exclusively a liquid phase protein sequencer. The N-terminal sequence (49 residues) was determined by degradation of the reduced and S-carboxymethylated protein. Other positions, i.e., residues 50-64, were identified by sequencing some tryptic fragments.

Primary structure of toxin IV of Leiurus quinquestriatus quinquestriatus: characterization of a new group of scorpion toxins

We report the sequence of toxin IV of Leiurus quinquestriatus quinquestriatus, one of the five toxins lethal for the mouse purified from the venom of this scorpion. Automatic sequencing of the S‐carboxymethylated protein and derived peptides obtained by action of the Staphylococcus aureus protease allowed us to work out the complete sequence of 65 residues. The toxin which was found to be blocked at the C‐terminal end has an extra residue at the N‐terminus as compared with other toxins from Asian and African scorpions. Moreover, toxin IV must be classified as an α‐scorpion toxin since it was shown to displace competitively 125I‐toxin II of Androctonus australis Hector (125I‐AaH II) from its binding site on rat brain synaptosomes. Nevertheless, it cannot be classified in one of the groups of scorpion toxins previously defined on the level of their antigenic properties. Again the classifications of scorpion toxins based on sequence homologies or antigenic properties support one another.We report the sequence of toxin IV of Leiurus quinquestriatus quinquestriatus, one of the five toxins lethal for the mouse purified from the venom of this scorpion. Automatic sequencing of the S-carboxymethylated protein and derived peptides obtained by action of the Staphylococcus aureus protease allowed us to work out the complete sequence of 65 residues. The toxin which was found to be blocked at the C-terminal end has an extra residue at the N-terminus as compared with other toxins from Asian and African scorpions. Moreover, toxin IV must be classified as an α-scorpion toxin since it was shown to displace competitively 125I-toxin II of Androctonus australis Hector (125I-AaH II) from its binding site on rat brain synaptosomes. Nevertheless, it cannot be classified in one of the groups of scorpion toxins previously defined on the level of their antigenic properties. Again the classifications of scorpion toxins based on sequence homologies or antigenic properties support one another.

Purification of animal neurotoxins: isolation and characterization of three neurotoxins from the venom of Naja nigricollis mossambica peters.

This work is part of a general study on snake neurotoxins. In previous papers [1,2], we had shown that the application to snake venoms of the general method of purification which was developed to isolate toxins from scorpion venoms [3-7] was very efficient. Four neurotoxins from two different sources ofNaja haje venom [1] and two neurotoxins fromNaja nigricollis venom [2] were purified when only one toxin had been previously isolated in a pure form from each of these venoms by others [8,9]. N-terminal sequences determination using automatic Edman degradation, was found to be the best method for rapid characterization of newly purified toxins [2]. We report, in this paper, on the application of these methods to the venom of Naja nigricollis mossambica Peters. The partial sequences of the neurotoxins show that these are more closely related to cobrotoxin (a toxin isolated from the venom of the formosan Elapidae Naja naja atra) than the toxins I and II isolated from the venom ofNaja nigricollis living in Ethiopia [2]. The taxonomical importance of these results is discussed.

Use of parvalbumin as a protecting protein in the sequenator: An easy and efficient way for sequencing small amounts of peptides

Since the description of the first automated apparatus for degradation of proteins by the phenylisothiocyanate method [l] it became obvious that the procedures worked out by Edman and Begg and later by Hermodson et al. [2] were not directly applicable to small and hydrophobic peptides. It has been shown that this was due to losses of the residual peptide during the extraction steps with organic solvents [ 1,3-61. Several alternative approaches have been developed to overcome the problem. One of these is to render the peptide suitable for the usual procedures by coupling polar groups either to lysine [4 ] and S-@-aminoethyl) cysteine residues [7] or to the C-terminal amino acid [.5,8]. A second approach is to adapt the procedure to the peptide either by decreasing the concentration of quadrol [6,8,9] or by using a volatile buffer [8] which, in both cases, limits the number and the volumes of organic solvents used for extraction. A third possibility is to attach the peptide to an insoluble resin and perform the degradation in an heterogeneous phase [lo] with the help of a specially designed apparatus [ 111. We wish to report the results we obtained in sequencing unmodified peptides in very small amounts (down to 20 nmol) in an Edman and Begg type sequenator using dimethylbenzylamine as coupling buffer but in the presence of a naturally aNHz-blocked protein (hake major parvalbumin) that acts as a protecting film. The technique proved also to be very efficient for the degradation of peptides attached to polystyrene resins: it prevents the loss

Utilization of aspirin, quinine and verapamil in the prevention and treatment of scorpion venom intoxication

Aside from serotherapy, the treatment of scorpion venom intoxication is symptomatic. The aim of this study was to compare the efficacy of drugs usually used in scorpion venom intoxications (atropine, propranolol) to that of other compounds, chosen in light of the pathophysiology of scorpion venom intoxication: dipyridamole, doxapram, quinine formate, lysine-acetyl-salicylate, valproate and verapamil. Using mice, the parameters evaluated were the preventive and therapeutic effects of drugs during experimental venom intoxication by Androctonus australis Hector and one of its toxins AaH 1, and by Buthus occitanus and Tityus serrulatus tunetanus on the other hand. It was found that although most of the drugs used could prolong the survival of the animals, the administration of verapamil and more so that of aspirin or quinine formate led to a 50 to 100% loss of venom and toxin toxicity, depending on the drug and the origin of the venom. In the case of propranolol, doxapram, atropine, dipyridamole and valproate, no or little protection were observed. If these results are confirmed in humans, the systematic use of these drugs could be a simple means for treating scorpion venom intoxication. The problem of scorpion venom intoxication poses a health problem both in the North African Maghreb and in the Americas. As a result of considerable information campaigns, the number of scorpion venom intoxications in Tunisia has dropped from 3000 in 1967 to 1000 per year in the 1980s. Serotherapy has reduced mortality to 0.35%, most deaths occurring in underweight children. In light of the large number of countries in which there is a risk of scorpion venom intoxication in the summertime, however, its prevention and treatment remain a major problem.