Purification and characterization of new antibacterial peptide from the Callinectes danae whole hemolymphy
Toyama, D.O.1, Reigada, A.2, Zara, J.F.2, Beriam, L.O.S.3, Toyama, M.H.2.
1Mackenzie, Universidade Presbeteriana Mackenzie, São Paulo, São Paulo, Brazil
2UNESP, Campus do Litoral Paulista, São Vicente, São Paulo, Brazil
3Instituto biológico de Campinas, Campinas, São Paulo, Brazil.
In this work we isolated a new antibacterial peptide from the whole hemolymphy extract from the Callinects danae. The whole hemolymph was lyophilized and subjected to HPLC molecular exclusion for first fractionation of crude hemolymph. After this first purification step the antibacterial fraction was subjected to new chromatographic step on the anion exchange column (Protein Pack SP 5PW) and some of these antibacterial fractions were subjected to new reverse phase purification. After this fractionation step, we obtained the antibacterial fraction named as CdIV-66, which was characterized as a basic and low molecular weigh protein from the whole hemolymphy of Callinectes danae crab. Its molecular mass was estimated by Tricine PAGE-SDS at 8 kDa and its amino acid sequence was determined as KILTQLWCKNSSVTYCCGQPPVWRPPFTSCVRRSGICVVIRSNIWQRCHI and showed significative amino acid similarities with other antibacterial peptide. This peptide decreases the bacterial growth rate of Xanthomonas axonopodis pv passiflorae (Gram negative), Clavibacter michiganensis michiganensis (Gram positive) and both case, this peptide induced several membrane damage and rupture of the cell wall. The CdIV-66 analysis showed that peptide has a random coil based structure with low content of alpha or beta structure. The electrophoretical analysis of hemolymph of recent collected and captured species was evaluated and our results suggest that stressing condition is involved in the highly increase production of these proteins.
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