Poster 187 - Bradykinin potentiating peptides in Lachesis muta snake venom: Identification of

Poster 187. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.546.

Bradykinin potentiating peptides in Lachesis muta snake venom: Identification of new peptides using mass spectrometry technologies and cDNA cloning.

Oliveira-Carvalho, A.L.¹; Wermelinger, L.S.¹; Soares, M.S.²;Dutra, D.L.S.¹; Bloch Jr.,C.³; Junqueira-de-Azevedo, I.L.M.⁴; Diniz, M.R.V.⁵; Ho, P.L.⁴ and Zingali, R.B.¹

1 Dep. Bioq. Medica, ICB, CCS, UFRJ. RJ, 2 IBCCF, CCS, UFRJ. Rede Proteômica do Rio de Janeiro, ³LNLS, Campinas-SP. ⁴Centro de Biotecnologia, Instituto Butantan. ⁵FUNED- MG Brasil.

Lachesismuta (Bushmaster) is the largest Crotalinae snake in the world. It is distributed in the tropical rain forests of Central and South America, including Brazil. As a consequence of envenomation, there are several predominant symptoms: intense local pain, nausea, abdominal colic, diarrhea, edema, hemostatic abnormalities, hypotension and neurotoxic signs. Most of this signs are due to the presence of proteins and peptides in the venom. One important application of MALDI TOF/MS is the direct analysis of complex samples allowing identification of low mass peptides and small proteins in a single step. In fact, this tool has been successfully used for the identification of a great number of peptides in crude venoms. The aim of this work is to use the MALDI-TOF/MS approach to obtain a mass pattern of the major peptides (<10 kDa) present in Lachesismuta crude venom. The samples were analyzed, using CHCA and SA matrix in the mass range 800-10.000 m/z with the linear mode. We identify in the MS spectra several peptides with molecular mass < 2000 Da and only one with 2211 m/z. Some of these peaks were submitted to MALDI TOF/TOF and de novo sequencing was preformed by precursor ion fragmentation using N2 and/or air collision to induced dissociation. Collision cell pressure was kept at 2.8 x 10^–6 torr. The sequence of four peptides was determined and shows high similarity with the BPPs (bradykinin potentiator peptides) described in the others snake venoms as B. jararaca and B. jararacussu. Parallel cDNA library sequencing was performed and resulted in the identification of the BPP precursor, of 1700 pb, highly expressed and encoding also the natriuretic peptide. Comparing the peptide masses from MALDI TOF, four of them matched with peptides deduced from the precursor and at least three of the derived sequences obtained by MALDI TOF/TOF perfectly matched the BPP precursor sequence. The combined proteomic and genomic represents powerful approaches that identified BPP peptides in the Lachesismuta venom, some of them being new uncharacterized ones.