Purification and Initial Biochemical Characterization of a Phospholipase A2
from the Social Wasp Polybia occidentalis Venom
Diniz-Sousa, R.1; Kayano, A.M.1; Coutinho-Neto, A.1; Moura, A.A.1; Monteiro,
M.C.2; Fuly, A.L.3; Calderon, L.A1,4; Soares, A.M.1,4; Stábeli, R.G. 1,4
1
Centro de Estudos de Biomoléculas Aplicadas à Medicina, CEBio, UNIR e
Fiocruz Rondônia, Porto Velho-RO; 2 Faculdade de Farmácia, Instituto de
Ciências da Saúde, Universidade Federal do Pará/UFPA, Belém-PA;
3
Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade
Federal Fluminense, UFF, Niterói-RJ; 4Fundação Oswaldo Cruz Rondônia,
FIOCRUZ, Porto Velho-RO, Brazil.
Venoms of hymenoptera (ants, bees and wasps) are constituted of a wide variety
of biologically active compounds such as peptides and proteins. Phospholipases
(PLAs) previously identified in venoms from Polybia genus hydrolysis
phospholipids from cell membrane, leading to pore formation, cell lyses and
tissues damage producing inflammatory reactions. This study aims the isolation of
a PLA2 from the venom of the social wasp Polybia occidentalis (Polistinae,
Vespidae). Crude venom of P. occidentails is able to hydrolyze fluorescent specific
PLA2 substrates (NBD-phospholipids). A higher and concentration dependent
PLA2 activity was observed toward NBD-Phosphatidylcholine instead of
phosphatidylglycerol or phosphatidic acid substrates. The PLA2 activity of the
crude venom was inhibited by EDTA (10 mM), suggesting a dependence of
divalent cations, possibly Ca2+. The venom was fractioned by a cation exchange
chromatography using a CM-Sepharose FF with sodium acetate 50 mM pH 5.2 at
1mL/min. resulting in five fractions. The last fraction (5th) showed PLA2 activity
against the substrate 4-nitro-3-octanoyloxy-benzoic acid. The 5th fraction was
pooled and submitted to a reverse phase chromatography using a Discovery C18
column and eluted with an increasing acetonitrile gradient. In order to determine
the purity and molecular mass of the phospholipase A2, 1D and 2D SDS-PAGE
were performed, showing a protein with mass approximately to 30 kDa.
Additionally, proteins with mass of 23 to 80 kDa were observed. Further assays in
order to obtain the N-terminal sequence of the protein are in progress. This study
was authorized by CGEN/CNPq (010627/2011-1) and IBAMA (27131-2).
Key Words: Hymenoptera venom, Phospholipase A2, Polybia occidentalis.
Supported by: FIOCRUZ, FINEP, CNPq, CAPES and Inpetam/INCT