PURIFICATION AND CYTOTOXIC CHARACTERIZATION IN MYOBLASTS AND MYOTUBES (C2C12) OF THE NEW FACTOR FROM Tityus serrulatus SCORPION VENOM

PONCE-SOTO L.A. (1), NOVELLO J.C. (1), MARANGONI S. (1)

(1) Department of Biochemistry, Institute of Biology, State University of Campinas, P.O. Box 6109, Zip code 13083 970, Campinas, SP, Brazil.

The new factor protein, was purified from Tityus serrulatus scorpion venom after one chromatographic step, reverse phase HPLC on m-Bondapack C-18. The molecular mass by SDS-PAGE was 4325.34 Da and confirmed by MALDI-TOF mass spectrometry .The amino acid composition showed that new factor have a high content of Lys, Asx, Gly, and 8 half-Cys residues, typical of a basic toxins from scorpions. Cytotoxic activity of new factor from Tityus serrulatus scorpion venom was assayed on murine skeletal muscle C2C12 myoblasts and myotubes. Variable amounts of toxin were diluted in assay medium (Dulbecco´s Modified Eagle´s Medium supplemented with 1% fetal calf serum) and added to cells in 96 well plates, in 150 ml. Controls for 0 and 100% toxicity consisted of assay medium, and 0.1% Triton X-100, respectively. After 3 h at 37 ºC, a supernatant aliquot was collected for determination of lactic dehydrogenase (LDH; EC 1.1.1.27) activity released from damaged cells, using a colorimetric end-point assay(Sigma 500C). Experiments were carried out in triplicate. The whole venom in vitro, did not lyse skeletal muscle myoblasts and myotubes, with a dose of 1.0 µg/well (1.0 µg/150 µl), in contrast the new factor lysed skeletal muscle myoblasts and myotubes, with a dose of 1.0 µg/well (1.0 µg/150 µl) causing 100% cytotoxic suggesting that the cytotoxic action of venom is strongly influenced by the new factor. The new factor displays a more restricted cytotoxic profile on cells in culture, mainly affecting differentiated skeletal muscle myotubes, which are cytotoxic on differentiated myotubes most but that in on myoblasts. Interestingly, the new factor, exerts a wider cytotoxic effect, suggesting an interaction with widely distributed targets in the membranes of other cells.

KEY WORDS: Tityus serrulatus, HPLC, Myoblasts, Myotubes and Cytotoxicity.

FINANCIAL SUPPORT: CAPES, FAPESP, CNPq

CORRESPONDENCE TO: Ponce-Soto, Luis Alberto Phone: + 19 35216132. Email: poncesoto@yahoo.com.ar