Molecular cloning and biochemical characterization of the first Na(+)-channel α-type toxin peptide (Acra4) from Androctonus crassicauda scorpion venom.

Due to the medical importance played in Turkey by stings of the scorpion Androctonus crassicauda, its venom has been studied with more attention. In this communication we report a new toxic peptide, named Acra4, because it is the fourth peptide completely characterized from venom of this scorpion. The peptide contains 64 amino acid residues stabilized by four disulfide bridges, with a molecular weight of 6937 Da. Purification of the lethal peptide was performed by three steps of high performance liquid chromatography (HPLC) separations, and the molecular weight was determined by mass spectrometry analysis and the full amino acid sequence was obtained by direct Edman degradation in conjunction with gene cloning. The LD50 of Acra4 was 50.5 ng/20 g mouse body weight (95% confidence intervals from 48.8 to 52.2 ng/20 g mouse body weight). Additionally, from a sample of cDNA of A. crassicauda four genes were cloned displaying sequence similarities to known scorpion toxins, and are reported here as potentially toxic peptides, named Acra5 to Acra8. Electrophysiological studies of Acra4 were performed using Na(+)-channels expressed in F11 cell culture, by patch-clamp recordings. This is the first time that such peptide from A. crassicauda having a specific Na(+)-channel α-type effect is reported. Its affinity toward Na(+)-channels in F11 cell line is in the order of 1 μM concentration.