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 M_r ∼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.