IDENTIFICATION OF CONTINUOUS INTERACTION SITES IN PLA2-COMPLEXES BY PEPTIDE ARRAYS

CONSUELO LATORRE FORTES-DIAS(1)

(1)Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG, Brazil

Crotoxin, the main neurotoxin of Crotalus durissus terrificus snake venom, is a heterodimer formed by an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA2 counterpart (CB). The SPOT synthesis methodology was used to map possible interaction sites in two PLA2-based complexes: CA.CB (crotoxin) and CNF.CB, a nontoxic complex formed between CB and an endogenous PLA2 inhibitor from C. d. terrificus plasma. Sets of overlapping dodecapeptides, spanning the whole primary structure of two main isoforms of CB (CB1 and CB2) were immobilized on a membrane and probed with CA or CNF previously conjugated to alkaline phosphatase. Main reacting peptide clusters were identified on the CBs as well as amino acid stretches susceptible to be involved in protein-protein interactions in the heterodimers. A highly reactive C-terminal cluster, exclusive to CB1, might explain, at least partially, the differences in stability of crotoxin, depending on the CB isoform present, as well as the apparent preference of CNF for CB2.

KEY WORDS: phospholipase A2 inhibitor, crotoxin, phospholipase A2, SPOT

synthesis, Crotalus durissus terrificus

CORRESPONDENCE: R. Conde Pereira Carneiro 80, 30510-010, Belo Hte, MG. Phone: +55 31 33719485; e-mail: consuelo@funed.mg.gov.br