Caracterização estrutural e farmacológica de peptídeos isolados do veneno de Bothrops bilineatus smaragdinus (Víbora Amazônica) com potencial atividade moduladora da liberação sináptica motora

Abstract

Bothrops bilineatus smaragdinus is an arboreal snake from the Amazon region whose toxinology has been negligibly studied. In this work, we proposed to purify and structural and pharmacologically identify presynaptic neurotoxins from B. b. smaragdinus venom with the potential modulating activity of motor synaptic release. The crude venom was subjected to gel filtration, with the fraction of interest then purified by RP-HPLC, and the resulting toxins were analyzed for their neurotoxic profile in a mouse phrenic nerve-diaphragm (PND) preparation in vitro. Toxins with excitatory function on motor neurotransmission were subjected to mass spectrometry to determine intact mass. Gel filtration revealed the existence of eight fractions (Peaks: P1–P8), with fraction P8 showing a potent concentration-dependent neuromuscular facilitating effect, without exhibiting phospholipase A2 (PLA2) activity. Fraction P8 (15.6 g/ml) produced a significant increase in the frequency of miniature endplate potentials (MEPPs). In addition, fraction P8 (15.6 µg/ml) significantly increased the amplitude of evoked endplate potentials (EPPs). Analysis by RP-HPLC and mass spectrometry of the P8 fraction determined the presence of two peptides with 652.4849 Da (P8-1) and 531.4108 Da (P8-2) of intact mass. The P8-1 peptide mostly exhibited the neuromuscular action seen with the P8 fraction, increasing the frequency of MEPPs and amplitude of EPPs in mouse PND preparations. These findings indicate that the P8-1 peptide is primarily involved in the neuromuscular excitatory effect previously seen with B. b. smaragdinus in mouse PND preparation, probably stimulating the release of acetylcholine from the motor nerve terminal.