Isolation, purification and pharmalogical and biochemical characterization of a new PLA2 (Bp12) from Bothrops pauloensis venom / Isolamento, purificação e caracterização bioquimica e farmacologica de uma nova fosfolipase A2 (Bp12) do veneno de Bothrops pauloensis

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

The myotoxic effects caused by bothropics venoms are unquestionable by their exuberant clinic manifestation and are extensively related in literature, since the neurotoxic effects have been described in vitro conditions, in nerve-muscle preparations, with little or neither clinic evidence. The aim of this study was to contribute for the knowledge about the pharmacological and biochemical characterization of Bp-12, a new myotoxin from B. pauloensis snake venom. The elution profile of B. pauloensis crude venom after purification in one chromatographic step presented eighteen peaks ranging from Bp-1 to Bp-18. Peak 12 was named Bp-12 and resulted in one small peak with a retention time of 37 min, eluting at 56% of buffer B. The SDS-PAGE gel showed one electrophoretic band, indicating that this toxin was obtained with high homogeneity and a molecular mass of around 14 kDa, confirmed by MALDI-TOF mass spectrometry, which showed that the protein had a molecular mass of 13789.56 Da. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA2 from other bothropics venoms. In mouse phrenic nerve-diaphragm (PND), the time needed for 50% paralysis was 17 ± 7 min (3.6 μM). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca2+ is replaced by Sr2+ or low temperature (22 °C), being the addition of d-tubocurarine required for direct blocking. The resting membrane potential showed that after 15 min of incubation with Bp-12 (3.6 μM), progressive despolarization decreased from –83.9 ± 1 mV to –20.2 ± 1 mV (90 min, p<0.05). Intracellular recordings of endplate potentials (EPPs) from mouse diaphragm preparations revealed that Bp-12 did no alter the quantal content (50 ± 8, t60, p>0.05 of the control). Incubation with Bp-12 (50 μg/mL) or B. pauloensis (50 μg/mL) for 120 min damaged 26 ± 2 % and 27 ± 1 % (p<0,05), respectively. Bp-12 (20 and 50 μg/50 μL) has significantly increased the release of CK levels in the serum after 60 min of i.m. administration, being the values: 827 ± 92 U/L and 225 ± 36 U/L (20 μg/mL). However, i.v. administration has not presented significant increased. Bp-12 (2.5 – 10.0 μg/paw) induced local oedema formation in rat paw in vivo at around 30 min, being found the values of: 0.4 ± 0.02 mL (2.5 μg/paw), 0.6 ± 0.05 mL (5.0 μg/paw) and 0.7 ± 0.07 mL (10.0 μg/paw). In conclusion, Bp-12 is a new myotoxic PLA2 (Lys49), with low catalitic activity, that is able to induce irreversible neuromuscular blockade and presents biochemical and pharmacological activities characteristic of bothropics miotoxins.

ASSUNTO(S)

bothrops neuromuscular junction eletrofisiologia eletrophysiology junção neuromuscular bothrops

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