ISOLATION OF A NEW BASIC PLA₂ FROM Bothrops jararacussu. PARTIAL BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION

BONFIM, V.L.; TOYAMA, M.H.; PONCE-SOTO, L.A.; OLIVEIRA, D.G.; NOVELLO, J.C.; MARANGONI, S.

Department of Biochemistry, Institute of Biology, UNICAMP, BRAZIL.

Objectives: In this work, we isolated and characterized a basic PLA₂ from the BthTX-II and present partial biological and biochemical characterization of this isoform. Methods and Results: The Bothrops jararacussu venom was fractionated on the HPLC ion-exchange chromatography (Protein Pack SP 5PW-Waters). The sample Bj-V (BthTX-II isoform) was subjected to reversed phase HPLC to further purification. The Bj-V showed allosteric enzymatic behavior with a non-micellar substrate, especially at low concentrations. Maximum catalytic activity occurs at a temperature of 35-40^oC and at pH 7,5-8,3. Full PLA₂ activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺; Cu²⁺ and Mg²⁺ inhibited this isoform when in the presence and absence of Ca²⁺, respectively. The enzymatic activity of Bj-V was significantly inhibited by differently crotapotins from rattlesnakes venoms. The Bj-V inhibited 92% of the growth of Clavibacter michiganensis sp michiganensis colonies. The N-terminal sequence (DLWQWGQMILKETGKLP...) showed a high degree of homology with basic D49 PLA₂ myotoxins and amino acid analysis proved a high content of hydrophobic and basic amino acids, as well as 14 half-cysteine  residues. Conclusion:The ion-exchange HPLC and RP-HPLC allow the purification of a new basic PLA₂ isoform (Bj-V) from the BthTX-II fraction. This novel enzyme showed a allosteric behavior similar to the one found in  Bj-IV (BthTX-II isoform too) but the N-terminal sequence showed slight differences between both isoforms, suggesting a minor differences in its structure. The venom from Bothrops jararacussu showed high antibacterial activity and isolated fraction inhibited 92% of the growth of Clavibacter michiganensis sp michiganensis bacteria.

Financial support: FAPESP

CORRESPONDENCE TO:

Vera Lucia Bonfim, Frei São Carlos 66, Campinas, SP, CEP: 13080-061, Brasil, Email: veralb@yahoo.com