Biochemical, Pharmacological, and Structural Characterization of New Basic Bbil-TX fromBothriopsis bilineataSnake Venom

Abstract

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25–37°C. Maximum PLA2 activity required Ca2+ and in the presence of Cd2+, Zn2+, Mn2+, and Mg2+ it was reduced in the presence or absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.

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

“…The PhTX-II PLA 2 is an enzyme composed of a unique polypeptidic chain revealed by Tricine SDS-PAGE with molecular mass of 14,149.08 Da confirmed by mass spectrometry ( Figure 2 ). This value is similar to other isolated myotoxic PLA 2 from snake venom [ 20 , 21 , 22 ].…”

“…Highest enzymatic activity occurs at pH 8 ( Figure 6 C). At low concentrations of substrate, PhTX-II showed a discrete sigmoidal behavior, as well as BbTX-III and Bbil-TX (purified from Bothrops brazili and Bothriopsis bilineata , respectively) that showed similar behavior towards this non-micellar substrate [ 17 , 21 ]. We can suggest that the specificity of the enzyme/substrate interaction for PhTX-II pointed to a structural resemblance of this enzyme with those PLA 2 that also had similar behavior with the substrate mentioned above.…”

“…The Asp49 PLA 2 myotoxins, besides causing muscle necrosis, have been shown to promote marked local inflammatory events in several experimental models [ 21 , 43 , 44 ]. The sub plantar injection of this PhTX-II PLA 2 also caused mouse paw edema ( Figure 8 B).…”

Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2

“…(Oliveira et al, 2008) and induced cytotoxicity in a primary cell culture of rat hippocampal neurons (de Carvalho et al, 2014). Many venom PLA2s have been shown to induce inflammation characterized by increase of vascular permeability (Zuliani et al, 2005a, Corasolla Carregari et al, 2013, edema formation (Ferreira et al, 2013, Kumar et al, 2015, and recruitment and activation of leukocytes (Zuliani et al, 2005b, Moreira et al, 2011.…”

Canopy Venom: Proteomic Comparison among New World Arboreal Pit-Viper Venoms

“…While investigations into Bothriechis , in particular B . schlegelii , venom [ 31 , 105 , 106 , 107 ] and some of the smaller bothropoid pitvipers [ 108 , 109 , 110 , 111 ] have occurred, knowledge gaps remain on the proteomic make up of the venom of the majority of New World pitviper species. This knowledge gap is unfortunate as snake venoms have proved valuable sources of investigational ligands [ 112 , 113 , 114 , 115 , 116 ] and potential therapeutics [ 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 ] due to the high degree of target specificity exhibited by their constituent toxins.…”