ISOLATION AND CHARACTERIZATION OF NEW BRADYKININ RELATED PEPTIDES FROM THE SKIN SECRETIONS OF PHYLLOMEDUSA HYPOCHONDRIALIS

CONCEIÇÃO K. (1), KONNO K. (1), MELO R.L.(1), ANTONIAZZI M.M. (2), JARED C. (2), LOPES-FERREIRA M. (1), PIMENTA D.C.(1).

(1) LETA – CAT/CEPID; (2) Dept. Biologia Celular, Instituto Butantan, São Paulo, SP, Brazil.

The amphibian skin secretions may provide new molecules with biotechnological applications, which should play different roles, either in the regulation of physiological functions of the skin or in the defense against predators or microorganisms. We targeted the identification of novel peptides with bradykinin-like structure present in the skin of this frog. Glandular secretions of P. hypochondrialis specimens were purified by rp-HPLC after solid phase extraction. MS fingerprinting was performed by MALDI-MS. Peptides were de novo sequenced on a ESI-Q-TOF-MS/MS. Peptides were synthesized by employing the Fmoc-strategy. BK activity was assessed by guinea-pig ileum contraction and by intravital microscopy. First, the SPE-processed sample was fractionated into 29 pools. The fractions were monitored by MALDI-ToF mass spectrometry for evaluation of molecular masses and purification grade. These pools were screened for low molecular weight peptides (700 to 1100 Da) with the aid of ESI-Q-TOF mass spectrometry. Several peptides were sequenced and three of them presented sequences that slightly resemble that of Bradykinin. These peptides were sequenced and identified as KPLWRL-NH2 (Phypo 3), RPLSWLPK (Phypo 5) and VPPKGVSM (Phypo 7a). Bradykinin-like peptides were chosen for the direct administration of the crude skin secretion cause several effects on the circulatory system, including inflammation.

KEY WORDS: Amphibian; Bradykinin; Phyllomedusa hypochondrialis

SUPPORTED BY: CNPq, CAPES and FAPESP.

CORRESPONDENCE TO: dcpimenta@butantan.gov.br