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Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake

Department of Biochemistry, Institute of Biology, State University of Campinas, Campinas, SP, Brazil.
Toxicon (Impact Factor: 2.58). 07/2009; 54(6):818-27. DOI: 10.1016/j.toxicon.2009.06.008
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ABSTRACT We report the purification and biochemical/pharmacological characterization of two myotoxic PLA(2) (BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2)) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing M(r) approximately 14 kDa for both myotoxins, showing high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2), had a sequence of 121 amino acid residues. BbTX-II: [amino acid sequence: see text] with pI value 8.73. BbTX-III: [amino acid sequence: see text] with a pI value of 8.46. BbTX-III presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+). In vitro, BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2) caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In mice, BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2) induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA(2) homologue, occur in the absence of a measurable PLA(2) activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.

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    • "MTX-II was obtained by the fractionation of B. brazili venom on a CM- Sepharose column (2 cm × 20 cm) as previously described [26]. Both proteins have 121 amino acids as determined by mass spectrometry experiments [25] [26], and there are five differences between their primary sequences: i) Glu4Gln, Lys20Ala, Gln68Pro, Asn111Gly and Ala130Pro, respectively for BbTX-II and MTX-II. Crystallization experiments were performed using sparse-matrix method [27], as previously described [28]. "
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