title: Biochemical, pharmacological, and structural characterization of new basic PLA2 Bbil-TX from Bothriopsis bilineata snake 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 Ca(2+) and in the presence of Cd(2+), Zn(2+), Mn(2+), and Mg(2+) it was reduced in the presence or absence of Ca(2+). 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.
authors.affiliation: Department of Biochemistry, Institute of Biology (IB), Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil.
authors.forename: Victor
authors.initials: V
authors.lastname: Corasolla Carregari
authors.forename: Rafael
authors.initials: R
authors.lastname: Stuani Floriano
authors.forename: Lea
authors.initials: L
authors.lastname: Rodrigues-Simioni
authors.forename: Flavia V
authors.initials: FV
authors.lastname: Winck
authors.forename: Paulo Aparecido
authors.initials: PA
authors.lastname: Baldasso
authors.forename: Luis Alberto
authors.initials: LA
authors.lastname: Ponce-Soto
authors.forename: Sergio
authors.initials: S
authors.lastname: Marangoni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004487:Edema / Q000473:pathology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007249:Inflammation; D007375:Interleukin-1 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000737:chemistry* / Q000494:pharmacology; D054497:Phospholipases A2, Secretory / Q000737:chemistry* / Q000494:pharmacology; D030162:Reptilian Proteins / Q000737:chemistry* / Q000494:pharmacology; D012910:Snake Venoms / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D007375:Interleukin-1; D015850:Interleukin-6; D030162:Reptilian Proteins; D012910:Snake Venoms; D014409:Tumor Necrosis Factor-alpha; C584304:Bbil-TX protein, Bothriopsis bilineata; D054467:Phospholipases A2; D054497:Phospholipases A2, Secretory; D002118:Calcium