Phospholipases A2 (PLA2s) from
snake venoms comprise a group of 14-18 kDa
proteins, responsible for several toxic effects induced by the whole
venom. Considering this, studies aiming at the search for natural inhibitors of these
proteins are very important. The present work had as objectives the isolation and functional/structural characterization of a γ-type
phospholipase A2 inhibitor (PLI) from Bothrops jararacussu snake plasma, named γBjussuMIP. This acidic
glycoprotein was isolated in a high purity level through affinity chromatography on CNBr-
Sepharose 4B coupled with BthTXII, showing a pI ∼ 5.5 and molecular weight of 23,500 for the monomer (determined by SDS-PAGE), and 160,000 for the oligomer (determined by molecular exclusion chromatography on
Sephacryl S-200). The interaction between γBjussuMIP (MIP) and
Phospholipase A2 (PLA2) was confirmed using circular dichroism (CD) and emission fluorescence techniques. The helical content of the 1:1 molar mixture was higher than that calculated for the addition of the spectra of the unbound
proteins indicating binding. The emission fluorescence experiments pointed that Trp residues in PLA2 participate in
proteins interaction as blue shift of 4 nm was observed. The γBjussuMIP
cDNA, obtained by PCR of the liver of B. jararacussu snake, revealed 543 bp codifying for a mature
protein of 181
amino acid residues. Alignment of its amino acid sequence with those of other snake γPLIs showed 89-94% of similarity. γBjussuMIP mainly inhibited the pharmacological properties of Asp49 PLA2s, such as
phospholipase,
anticoagulant, myotoxic,
edema inducing, cytotoxic, bactericidal and lethal activities. In addition, it showed to be able to supplement Bothrops
antivenom, potentiating its antimyotoxic effect. The aspects broached in this work will be able to provide complementary information on possible mechanisms of action, relating structure and function, which could result in a better understanding of the inhibitory effects induced by γBjussuMIP.