Primary Structure and Physical-Chemical Properties of Alternatustoxin (BtTX), a PLA2 Lys49 from Bothrops alternatus (Urutu) Snake Venom and Its Neurotoxic Properties

¹Ponce-Soto, L.A.; ¹Fagundes F.H.R.; ¹Bonfim, V.L.; ¹Damico, D.C.S.; ²Rodrigues-Simioni, L. and ¹Marangoni. S.

1 Departament of Biochemistry Institute of Biology and 2 Departament of Pharmacology Faculty Medical Science UNICAMP – BRAZIL. 

Alternatustoxin (BtTX), PLA2 (K49) was purified from the of Bothrops alternatus venom after two chromatographic steps, molecular exclusion in a column Superdex G-75 and reverse phase HPLC system m-Bondapack C-18. The molecular mass were estimated by electrophoresis SDS-PAGE 13.5 kDa, and by MALDI-TOF mass spectrometry (13898.7121 Da) too the composition of amino acids showed that the fraction Alternatustoxin (BtTX), an Lys49 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues showing to be a basic PLA2. The complete amino acid sequence of Alternatustoxin (BtTX), contained 121 amino acid residues, with a primary structure of: SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. The amino acid sequence of the Alternatustoxin (BtTX), PLA2 (K49) revealed high sequence homology with other PLA2s. this results showed highly conserved amino acid sequences (95%) with other PLA2 K49 from origin botropic. This value diminishes when it is compared with other PLA2 D49. It presents two fundamental substitutions, Y for G(25) and W for V(30). The fraction Alternatustoxin (BtTX), caused a dose-dependent blockade of the neuromuscular transmission when using mouse phrenic nerve-diafragm preparation. The blocking effect was observed using 10 and 20 mg/ml. respectively (n=5/dose). These doses showed a total and irreversible twitch tension blockade. In the control preparations, the twitch tension records remained stable (93.9% all along the 120 min of incubation in Krebs solution, described by Bülbring (1946).

Financial Support: FAPESP and CAPES.

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