Structural studies of Bothroinsularin. a thrombin inhibitor protein fromBothropsinsularis venom

A L Oliveira-Carvalho, M. Assafim, D.L.S. Dutra, H.C. Castro, R.B. Zingali.

Departamento de Bioquímica Médica, ICB, CCS, UFRJ

Members of the protein family named C-type lectins have been purified of venoms  from Viperidae snakes. These proteins display high amino acid sequence similarity although  presenting diverse biological functions. Bothrojaracin (bjc), purified from  Bothrops jararaca, a thrombin-binding protein (27 KDa) is a member of this family. Western blots  analysis of Bothrops insularis crude venom show a  variety of bands recognized by the  polyclonal antibodies (anti-bjc), including a ~30 KDa protein. Methods and results: Purification of crude venom by gel filtration followed by affinity chromatography on a PPACK--thrombin sepharose resulted in a protein consisting  of a dimer  formed by  two 15 KDa chains as observed by SDS  PAGE under reducing condition. This protein was recognized  in western-blot assays  using polyclonal antibodies raised against bothojaracin.  Its N-terminal sequences (25aa), showed high similarity  with bothrojaracin (90%)  and others members of c-type lectin family. Bothroinsularin formed a complex with -thrombin with a molar rate of 1/1. Similar data was obtained with prothrombin. The carboxymethylated and subunits were separated by a reversed- phase chromatography (Sephasil C8 Pharmacia) in an HPLC System.

Conclusion and perspectives: Here we describe the purification and characterization of a thrombin-binding protein named bothroinsularin. This protein shows high similarity with bjc. The sequence of the separated chains will be determined for further cloning and expression.

Financial support: CNPq, CEE, Finep, Faperj

CORRESPONDENCE TO:

Ana Lucia de Oliveira Carvalho, Rua do Ouro 220, Duque de Caxias, RJ, CEP: 25050120, Brasil, Email: aloliveira@bioqmed.ufrj.br