purification and characterization of Antimicrobial peptides isolated from the CUTANEOUS secretion of THE FROG Leptodactylus ocellatus
Nascimento, A.C.1.; Fontes, W.2; Sousa, M.V.2; Kyaw, C.3; Schwartz, C.A.1; Sebben, A.1; Schwartz, E.F.1 and Castro, M.S.1,2
1Laboratório de Toxinologia, Departamento de Ciências Fisiológicas/IB, Universidade de Brasilia, Brasília/DF; 2CBSP - Centro Brasileiro de Serviços e Pesquisas em Proteínas, Departamento de Biologia Celular/IB, Universidade de Brasilia, Brasília/DF; Laboratório de Microbiologia, Departamento de Biologia Celular/IB, Universidade de Brasilia, Brasília/DF.
Antimicrobial peptides are remarkably found in the skin secretion of frogs and function as an initial barrier against infections. They can promptly kill microorganisms by damaging their cytoplasmatic membranes. Since such effect is not mediated by receptors, it is possible that peptide-based antibiotics can be used against drug-resistant pathogens without bringing about new resistance genes. The peptides produced by distinct frog species show differences that may lead to the discovery of novel bioactivities. In the light of this fact, the purification and characterization of antimicrobial peptides in the skin secretion of Leptodactylus ocellatus is reported in the present study.
Adult specimens of Leptodactylus ocellatus were collected in Distrito Federal region and kept in captivity. Cutaneous secretion was obtained by mild electrical stimulation and lyophilized. The dry secretion (5.0 mg) was dissolved in 0.1% (v/v) TFA/water and purified by RP-HPLC in C8 column. Elution was performed using a linear gradient of acetonitrile, at a flow rate of 0.8 mL/min. Absorbance was monitored at 216 nm. Fractions were manually collected and then lyophilized. Fractions of interest were purified to a higher degree by RP-HPLC in C18 column. Three abundant fractions had their molecular masses determined by m-ESIMS (API 300, Sciex-PE) and were sequenced by automated Edman degradation using an ABI 477A sequencer. The studied fractions have sequences of 20, 21 and 25 amino acids residues with amidated C-terminus, which is expected for amphibian antimicrobial peptides. Antimicrobial properties were assessed by antibacterial plate assay using Escherichia coli and all peptides exhibit antibacterial properties.
Financial support by: FINATEC and FUB/UnB