細胞科学と治療のジャーナル
オープンアクセス

概要

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

Frey Francisco Romero-Vargas, Thalita Rocha, Maria Alice Cruz-Höfling, Lea Rodrigues-Simioni, Luis Alberto Ponce-Soto and Sergio Marangoni

In this research a preliminary identification and biochemical and biological characterization of a PLA2 (Btae TX-I) from the venom of a viperid snake, Bothriopsis taeniata (Speckled forest pit viper) were obtained. Btae TX-I was purified by two chromatographic steps, molecular exclusion chromatography followed by analytical chromatography reverse phase HPLC. Molecular mass behaved as a homogeneous single chain protein on SDS–PAGE, confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 13889.98 Da. Tryptic peptides were determined in tandem mass spectrometry and showed similarity with other myotoxic PLA2s. Btae TX-I belongs to the Asp49 PLA2 class, is enzymatically active in presence of a synthetic substrate and shows a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35–45°C. PLA2 activity in presence of Mn2+, Mg2+, Cd2+ and Zn2+ was reduced either in presence or absence of Ca2+, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+. Crotalic crotapotins from rattlesnake venom has significantly inhibited (p<0.05) the enzymatic activity of Btae TX-I. In ex vivo experiment, Btae TX-I caused partial blockade of the neuromuscular transmission in chick biventer cervicis preparations in a similar way to other Bothrops species. Btae TX-I also inhibited contractures in the upper concentration (50 μg) to exogenous KCl (20 mM). Histological analysis of the biventer cervicis incubated with Btae TX-I showed that just the highest Btae TX-I PLA2 dose (50 μg) caused almost 27.4 ± 0.3% damaged fibers. The results give evidence that the main effect of type Asp49 Btae TX-I PLA2 from Bothriopsis taeniata is at the post-synaptic site.