Abstract
A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.
Highlights
IntroductionSnakes of the genus Bothrops (including Porthidium and Botriopsis) represent the ophidian fauna of great scientific and medical interest in Brazil, since they are responsible for most cases of deadly snake bites which occur in the country [1,2]
Snakes of the genus Bothrops represent the ophidian fauna of great scientific and medical interest in Brazil, since they are responsible for most cases of deadly snake bites which occur in the country [1,2]
Emphasized in black is fraction 11 (*) characterized as PhTX-II phospholipase A2 (PLA2); Insert: Electrophoretic profile in Tricine SDS-PAGE (1) Molecular mass markers; (2) PhTX-II not reduced; (3) PhTX-II reduced with DTT (1 M)
Summary
Snakes of the genus Bothrops (including Porthidium and Botriopsis) represent the ophidian fauna of great scientific and medical interest in Brazil, since they are responsible for most cases of deadly snake bites which occur in the country [1,2]. Minimum attention has been paid to the characterization of P. hyoprora venom. This snake is named Amazonian hog nose pit viper, whose venom has high phospholipase A2 activity and induces drastic local myotoxicity [6]. The phospholipase A2 (PLA2) superfamily consists of a broad range of enzymes defined by their ability to catalyze the hydrolysis of the middle (sn-2) ester bond of substrate phospholipids [7]. This family of proteins can be found in the mammalian pancreas and in the venoms of snakes, scorpions and bees. A PLA2 may have more than one specific physiological activity, and it may play multiple roles in the overall effects of envenoming [9]
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