PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry

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.

ACP-TX-I and ACP-TX-II, Two Novel Phospholipases A2 Isolated from Trans-Pecos Copperhead Agkistrodon contortrix pictigaster Venom: Biochemical and Functional Characterization

“…F5 and F6 crotapotins from C. d. collilineatus inhibited the enzymatic activity of ACP-TX-II PLA 2 by approximately 55% (Figure 4F). These results are consistent with PhTX-II PLA 2 from Porthidium hyoprora , which was inhibited ~60% by F2 and F3 crotapotins from C. d. collilineatus [8]. These results suggest that crotapotins may bind to Agkistrodon PLA 2 , much like how they bind to crotaline PLA 2 s.…”

“…Using this purification method, several other PLA 2 (Bp13 PLA 2 , PhTX-I, -II,-III, Bleu-PLA 2 , Bbil-TX, BbTX-II, -III, etc.) from different venoms have been purified, showing that it is rapid and efficient for the purification of these proteins in one step [6,7,8,35,36,37,38].…”

“…Output masses ranged from 6000–20,000 Da at a 0.1 Da/channel “resolution.” The simulated isotope pattern model was used with the spectrum blur width parameter set to 0.2 Da, and minimum intensity ratios between successive peaks were 20% (left and right). After the smoothing of deconvoluted spectra, mass centroid values were obtained using 80% of the peak top and a minimum peak width at half the height of 4 channels [8].…”

“…PLA 2 s are small proteins (13–15 kDa) with 115–122 residues, and seven conserved disulfide bonds [5,6]. They are subdivided into two main subgroups: (1) D49 PLA 2 s, which have an aspartate residue at position 49, and typically have high catalytic activity [7,8], and (2) K49 PLA 2 s, with a lysine residue at position 49. These have little or no catalytic activity, but still induce various biological effects [9,10].…”

Biological characterization of the Amazon coral Micrurus spixii snake venom: Isolation of a new neurotoxic phospholipase A2

“…Viperid PLA 2 s show a greater limitation in maintaining their phospholipasic activity when they are submitted to variations in pH and temperature, showing maximum activity only within the narrow range of 35 and 40 C and pHs between 7.0 and 8.0 (Heleno et al, 2013;Huancahuire-Vega et al, 2014). This limitation of activity in function of temperatures and pHs near physiological levels, seems to be characteristic of group II PLA 2 s (viperid) and it is also observed that human PLA 2 s lose stability and activity outside of this range (Leidy et al, 2006).…”