Poster 069 - Amino acid sequence of col F3 crotapotin isoform isolated from the Crotalus durissus

Poster 69. 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.428.

AMINO ACID SEQUENCE OF COL F3 CROTAPOTIN ISOFORM ISOLATED FROM THE Crotalus durissus collilineatus VENOM.

Toyama, D.O.¹, Toyama, M.H.², Câmara, P.R.S.³, Beriam, L.O.⁴, Camargo, E.A.³, Joazeiro, P.P.⁵, de Nucci, G.³, Antunes, E.³ and Marangoni, S.¹

1 Departamento de Bioquímica, IB, UNICAMP; 2 CLP, Unidade de São Vicente, UNESP; 3 Departamento de Farmacologia, FCM, UNICAMP; 4 Instituto Biologico, Campinas, 5 Departamento de Histologia e Embriologia, IB, UNICAMP.

In this work we determined the complete amino acid sequence of this novel crotapotin (Col F3) isolated from the Crotalusdurissus collilineatus as well as its biological activity. The purification of Col F3 acid subunit of the venom was isolated after the molecular exclusion and reverse phase HPLC, and the reduction and S-carboxymethylation of purifed acid subunit (Col F3) was done with guanidine chloride solution and DTT 6M and carboxymethyled with ¹⁴C-iodoacetic acid. The complete amino acid sequence of Col F3 was determined on the Procise f sequencer. The measurement of rat dorsal skin oedema was expressed as the volume (ml) of plasma accumulated at each skin site compared to total counts in 1 ml of plasma from the male Wistar rats. Insulin secretion from the isolated islets was measured by radioimmunoassay using rat insulin as the standard, in this protocols we used Col F3 native or treated with p-Bromophenacyl bromide (BPB). The antibacterial activity was done used fresh agar plate incubated with bacteria treated and no treated with crotaporin. The Col F3 is composed of three separate polypeptides identified as Chain A, B and C. The amino acid sequences of the Chain A and that of the enzymatically active PLA2 were quite similar in regarding to the calcium binding, catalytic site, B-wing and helix 3 regions. This crotapotin also showed significant edematogenic and insulin secretion activity, which was inhibited by incubation of this protein with BPB. This protein showed significant antibacterial effect against both bacterial strain (Xanthomonasaxonopodis pv. passiflorae and Clavibactermichiganensis subesp. michiganensis). IIn conclusion, these results suggested that this novel Crotapotin may be a potential tool for the understanding of the model of action of PLA2 and the role of the catalytic site of the PLA2 enzymatic active in the expression of biological action.