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The purification of eleven neurotoxins from the venom of three scorpions (Androctonus australis Hector, Buthus occitanus tunetanus and Leiurus quinquestriatus quinquestriatw8) has been performed by Sephadex G-50 filtration (with and without recycling) and equilibrium chromatography on Amberlite CG-50, CM-Sephadex C-50 and DEAE-Sephadex A-50. All the chromatographic steps were carried out in ammonium acetate buffers. Purity of the neurotoxins was assessed by amino acid analysis and zone electrophoresis. All have a molecular weight of about 7000 daltons and do not contain methionine. As shown by amino acid composition and chain length scorpion neurotoxins show a high degree of homology.From the first attempts made in this laboratory to purify the neurotoxins of scorpion venoms, successive improvements in the purification method have been introduced. Snake or scorpion neurotoxins are basic proteins of small molecular weight (about 7000). Two properties of these neurotoxins, solubility in pure water [l] and basicity as shown by zone electrophoresis [2], served as guide-lines to use reversible adsorption on Sephadex G-25 swollen in water [3] and ion-exchange chromatography on Amberlite IRC-50 in a volatile buffer a t near neutral pH, to obtain the purified neurotoxins [2,4,5]. Despite homogeneity in the ultracentrifuge, by starch gel electrophoresis and by equilibrium chromatography on Amberlite IRC-50, further studies have shown the purified proteins to be dimers of non-covalently associated monomers or non-covalent association of neurotoxins with non-toxic material Two pure monomeric neurotoxins from the NorthAfrican scorpion Androctonus australis Hector collected in the area of Tozeur (Tunisia) [ill were later obtained with Sephadex G-50 filtration, chromatography on Amberlite CG-50 a t pH 6.30 and 6.70 and on DEAE-Sephadex A-50 a t alkaline p H (8.50) still in volatile buffer.This and the following paper describe a general method of purification of the neurotoxins contained in animal venoms (scorpions and snakes). Improvement over the previously described method [ll] was obtained by introducing recycling gel filtration on Sephadex and the use of another cation-exchanger, CM-Sephadex C-50. I n this first paper we describe the isolation and the characterization of eleven pure EXPERIMENTACPROCEDURE MATERIALS VenomsThe venoms of Buthus occitanw tunetanus (Mecheria, Algeria) and Leiurus quinquestriatus quinquestriatus (area of Khartum, Sudan) were obtained from F. G . Celo (Zweibriicken, Germany). The scorpions Androctonus australis Hector were collected in the area of Chellala (Algeria) by the Institut Pasteur of Algeria. They were maintained alive in our laboratory in individual plastic boxes and received food (mealworms) and water via soaked sponges each month. The animals were kept a t 22-25" except for the week preceding milking during which the temperature was raised to 30-33". Milking was obtained by cyclic electrical stimulations of the postabdomen (55 volts for 0.5 sec followed by a rest of the same duratio...

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The primary structure of toxin III of Leiurus quinquestriatus quinquestriatus (Lqq III) was elucidated by automatic Edman degradation of the reduced and S-carboxymethylated protein and derived tryptic peptides. Like other scorpion toxins that are active on sodium channels, Lqq III, consisting of 64 amino acids, is a 7 kDa single-chain polypeptide crosslinked by four disulfide bridges. It belongs to the alpha-toxin group, as judged by competition experiments with 125I AaH II for binding to rat brain synaptosomes (K0.5 = 7 x 10(-7) M). Lqq III is the first alpha-toxin to be characterized that is highly toxic to mice [LD50 = 50 micrograms (7.1 nmol)/kg body wt], by subcutaneous injection, insects Blatella germanica [LD50 = 60 ng (8.5 pmol)/g body wt.] and Musca domestica [LD50 = 120 ng (17 pmol)/g body wt]. When tested via the intracerebroventricular route, the toxicity for mice [55 micrograms (8 nmol)/kg] was of the same order as that found by subcutaneous injection, indicating that Lqq III has a higher affinity for peripheral sodium channels that for those of the central nervous system. There are three differences between the sequences of Lqq III and Lqh alpha IT, an alpha-toxin isolated from the venom of Leiurus quinquestriatus hebraeus. These substitutions are found at positions 20, 24, and 64 (Ser-->Ala,Asp-->Glu and His-->Arg, respectively). Surprisingly Lqh alpha IT is only weakly active in mice [LD50 = 5 mg (0.7 mumol)/kg], while in insects its toxicity is similar to that of Lqq III [140 ng (20 pmol)/g body wt blowfly larvae]. These observations are relevant to the definition of scorpion toxin structure-activity relationships.

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