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

We report the purification and biochemical/pharmacological characterization of two myotoxic PLA2 (BbTX-II K49 PLA2 homologue and BbTX-III PLA2) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing Mr ∼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 PLA2 homologue and BbTX-III PLA2, had a sequence of 121 amino acid residues. BbTX-II: SLFELGKMILQETGKNPAKSYGAYGCYCGVLGRGKPKDATDRCCYVHKCCYKLTGCDNKKKDRYSYSWKDKTIVCGENNPCLKELCECDKAVAICLRENLNTYNKKYRYHLKPLCKKADAC with pI value 8.73. BbTX-III: SLWEWGQMILKETGKNPFPYYGAYGCYCGWGGRRKPKDATDRCCFVHDCCRYKKLTGCPKTNDRYSYSRLDYTIVCGEDDPCKEICECDKAAAVCFRENLRTYNKKYMAHLRVLCKKDKPC with a pI value of 8.46. BbTX-III presented PLA2 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 °C. Maximum PLA2 activity required Ca2+. In vitro, BbTX-II K49 PLA2 homologue and BbTX-III PLA2 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 PLA2 homologue and BbTX-III PLA2 induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA2 homologue, occur in the absence of a measurable PLA2 activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.