Purification and structural characterization of a homodimeric peptide from skin secretion of Phyllomedusatarsius (Amphibia: Anura)

MORALES, R.A.V.(1,2), PRATES, M.V.(1), SANTOS, N.C.F.(1), BLOCH JR., C.(1)

(1)Embrapa, Recursos Genéticos e Biotecnologia, (2)Instituto de Ciências Biológicas, Universidade de Brasília, UnB

The amphibian skin contains granular glands that are responsible for the production of several classes of useful biotechnological compounds, such as alkaloids, opiod peptides and antimicrobial peptide. These compounds are usually associated with amphibian chemical defense against potential predators such as animals and microorganisms. In this work, we describe the structural characterization of a new homodimeric peptide found in the skin secretion of Phyllomedusa tarsius from Amazon rain forest.

The skin extracts were obtained by mild electrical stimulation, collected in Milli-Q water, and were later frozen, and lyophilized. The water-soluble fractions from the crude extract were fractionated by RP-HPLC, using a semi-preparative C₁₈ column in a linear acetonitrile gradient with 0.1% of trifluoracetic acid and monitored in 216nm and 280nm. The fraction of interest was manually collected and submitted to a further purification step using analytical C₁₈ column in the same conditions. The purified fraction was reduced with DTT, alkylated with iodoacetamide and had its primary structure obtained by Edman degradation in an automated system. The purity and molecular mass was analyzed in each characterization step by mass spectrometry using a MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time-of-Flight) system and a-cian-4-hydroxycinamic acid as matrix.

The analyzed compound show to be a homodimeric structure of 5509.40Da composed by two identical polypeptide chains of 24 amino acid residues with 2755.21Da each, linked by a single disulphide bond between the Cys23 of each chain. Structurally it is very similar to the heavy chain of Distintin, an efficient heterodimeric antimicrobial compound from Phyllomedusa distincta previously described by our group (Batista et. al. 2001, FEBS Letters 484:85-89)

CORRESPONDENCE TO:

MORALES, R.A.V., SHIS QI 17 Conjunto 03 casa 19, Brasília, DF, CEP: 71645030, Brasil, Email: moralesrodrigo@zipmail.com.br