PUTATIVE ANTIMICROBIAL PEPTIDES ISOLATED FROM THE SKIN SECRETION of THE FROG Leptodactylus ocellatus

Nascimento, A.C.(1), Fontes, W.(2), Sousa, M.V.(2), Schwartz, C.A.(1), Schwartz, E.F.(1), Sebben, A.(1), Castro, M.S.(1,2)

(1)Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, IB, Universidade de Brasília, (2)Laboratório de Bioquímica e Química de Proteínas, Centro Brasileiro de Serviços e Pesquisas em Proteínas, Departamento de Biologia Celular, IB, Universidade de Brasília, Brasília, DF, Brasil.

The exudate of frog skin contains an array of gene-encoded peptides with diverse biological functions. The main functions attributed to this repertoire of peptides are preventing microbial infection, assisting wound healing and helping to avoid predation. In the present report we describe the purification and characterization of two hemolytic peptides obtained from Leptodactylus ocellatus cutaneous secretion. Adult specimens of Leptodactylus ocellatus were collected in Distrito Federal region and maintained in captivity at the University of Brasília. Skin secretion was obtained by mild electrical stimulation and lyophilized. The dried secretion (5.0 mg) was dissolved in 0.1% (v/v) TFA/water and loaded onto a C₈ reversed-phase column (Sephasil Protein, 4.6 x 250 mm, Pharmacia Biotech).. Elution was performed using a linear gradient of acetonitrile, at a flow rate of 0.8 mL/min. The absorbance was monitored at 216 nm. Fractions were manually collected, lyophilized and tested for hemolytic activity. Two hemolytic fractions were individually rechromatographed using a C₁₈ reversed-phase column (Vydac 218TP54, 4.6 x 250 mm, Separations Group), yielding symmetric peaks. The purified toxins were analyzed by m-ESIMS (API 300, Sciex-PE) and submitted to automated Edman degradation using an ABI 477A sequencer, resulting in unambiguous sequences. A search using nonredundant database by BLASTP revealed homology to amphibian antimicrobial peptides included in the brevinins family.

Financial supported by: FUB, UnB and FINATEC.

CORRESPONDENCE TO:

Anna Christina Carvalho do Nascimento, SQS 205 - bloco B - Apto. 508, Brasília, DF, CEP: 70235020, Brasil, Email: malk@brturbo.com