Linear protein-protein interactions in crotoxin by spot synthesis: identification of contact sites between its subunits

SANTOS, R.M.M.(1), CHÁVEZ-OLÓRTEGUI, C.(1), GRANIER, C.(2), FORTES-DIAS, C.L.(1)

(1)Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG, (2)Faculté de Pharmacie, CNRS UMR 9921, Montpellier, France.

The venom from Crotalus durissus terrificus is composed mainly of a neurotoxic protein named crotoxin, which is known to contain the bulk of the lethal toxicity of the crude venom. Crotoxin is a heterodimer composed of a basic toxic PLA₂ subunit (CB) and of an acidic, non-toxic and non-enzymatic subunit (CA). In the present work we have used the SPOT method of multiple peptide synthesis to measure the ability of CA to bind to linear segments of CB. Sets of overlapping short peptides (12 residues) spanning the primary amino acid sequences of two main isoforms of CB were immobilized onto a cellulose membrane and probed with CA previously labelled with alkaline phosphatase. Colour intensities were measured after scanning the membrane and relative reactivities were calculated with reference to a black spot taken as 100 %. Four to five linear regions of CB have been mapped that might be involved in the interaction with CA in the crotoxin complex.

Financial support: FAPEMIG and CNPq

CORRESPONDENCE TO:

Consuelo Latorre Fortes-Dias, Rua José Mendes de Carvalho 250, Belo Horizonte, MG, CEP: 30840-350, Brasil, Email: consuelo@funed.mg.gov.br