TWO-DIMENSIONAL ELECTROPHORESIS APPLIED FOR SNAKE VENOM ANALYSIS AND FOR THE EVALUATION OF ITS IMMUNOGENICITY

Flores, M.P.A.(1), Ricci-Silva, M.E.(1), Furtado, M.F.D.(2), Rádis-Baptista, G.(1), Prieto da Silva, A.R.B.(3), Yamane, T.(1)

(1)Molecular Toxinology Lab., (2)Herpetology Lab., (3)Biotechnology Center, Institute Butantan, São Paulo, SP, Brazil

The two-dimensional electrophoresis (2-D PAGE) technique, first described by O'Farrell in 1975 (J.Biol.Chem. 250:4007), is the preferred method and still the highest resolution technique for gene product analysis. By combining conventional one-dimensional SDS-PAGE and isoelectric focusing (IEF) techniques, each with resolving power of -100 proteins, it can have a theoretical resolution of 10,000 individual spots. However, e.g., HeLa cells contain as many as 23,500 poly(A)-mRNA species, and many of them are low abundance proteins. Therefore, Proteomics faces a formidable challenge, far beyond our current technical abilities, unless the question is narrowed to a subset of proteins (vide Gavin, A., et at. Nature 415: 141-147, 2002) Venom glands of animals contain a limited number of peptides/proteins, normally less than 50, with antigenic properties, therefore, amenable to 2-D PAGE analysis. Anti-venom sera rose against a given snake venoms should contain a great number of antibodies, some of which are important for neutralizing the venom's lethal effect. Cross-reactivity is a very common phenomenon, i.e., snake venoms that were not used in immunization for the sera production, can be neutralized by immunosera raised against different snake venoms. This leads to the assumption that venom of different species of genra may present common antigens/epitopes. It is quite clear that 2-D PAGE is the most suitable for identifying cross-reacting toxins. Our studies on Bothrops jararaca venom by 2-D PAGE and subsequent identification of antigenic components using polyvalent anti-Bothrops sera have revealed the following; (a) pl of venom components lies between 4 and 7; (b) not all components are immunogenic and it is not correlated with lhe amount present as detected by silver staining; (c) among isoforms, those with a lower pl seem to be more antigenic.

Support: FAPESP

CORRESPONDENCE TO:

MARIA ESTHER RICCI DA SILVA, Avenida Vital Brasil 1500, Butantan, São Paulo, SP, CEP: 05503900, Brasil, Email: esthericci@hotmail.com