An α-type phospholipase A_2 inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

摘要

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A_2 from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated αBjussuMIP, showed it to be an oligomeric glycoprotein with M_r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% α-helix, 18% β-sheet, 10% β-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either αBjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of αBjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these αPLIs. aBjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 ℃ to 80 ℃, even after deglycosylation. The results showed effects against Bothrops phospholipase A_2 activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that αBjussuMIP may prove useful in the treatment of snakebite envenomations.