PURIFICATION AND CHARACTERIZATION OF PEPTIDE TOXINS FROM THE SEA ANEMONE Bunodosoma cangicum

Zaharenko, A.J.(1,2), Oliveira, J.S.(1,2), Freitas, J.C.(1,2), Portaro, F.C.V.(2), Pimenta, D.C.(2), Yamane, T.(2), Konno, K.(2)

(1)Departamento de Fisiologia, Instituto de Biociências, USP, São Paulo, Brazil, (2)Center for Applied Toxinology, CAT-CEPID, FAPESP, Instituto Butantan, São Paulo, Brazil.

AIM: To purify and identify neurotoxic and haemolytic compounds previously reported in the venom of the sea anemone Bunodosoma cangicum.

MATERIALS AND METHODS: The venom was obtained by electrical stimulation of the whole animals in artificial sea water. It was lyophilized and gel-filtered in a Sephadex G-50 column, yielding five peaks. The haemolytic fraction was detected in mouse erythrocyte suspension and the neurotoxic fraction assayed in crab leg nerve bioassay and further purified by RP-HPLC (214nm) in a Beckman C-18 (150mm X 4.6mm) analytical column performed in linear gradient from 5% to 50% of buffer B in 25min, revealing three major peaks. These peaks were analyzed by Q-TOF and MALDI-TOF mass spectroscopy. SDS-PAGE was also carried out with the haemolytic fraction in order to determine the approximate molecular weight of the cytolysins.

RESULTS: The cytolytic compounds showed molecular masses between 10kDa and 20kDa, suggesting that they belong to the typical class of anemone cytolysins. Two of the three major peaks obtained from HPLC showed strong neurotoxic activity and molecular masses of 4156Da and 4975Da. The former´s molecular mass is near to those of some known sea anemone K⁺ channel blockers (BgK: 4282Da and ShK: 4061Da). The latter is probably related to the long sea anemone peptides which are known to delay the inactivation of the voltage-gated Na⁺ channels. ATX I, II and V from Anemonia sulcata and cangitoxin from B. cangicum have molecular masses closely related to this compound.

CONCLUSIONS: We conclude that the polypeptidic mixture obtained directly from nematocyst discharge by electrical stimulation may provide us less levelsof the body contaminants with great amounts of neurotoxic and haemolytic compounds. Some of these fractions may prove to be novel K⁺ and Na⁺ channel toxins. We are currently determining the primary sequence of these neurotoxic peptides.

ACKNOWLEDGEMENTS: CEBIMar, USP and FAPESP.

CORRESPONDENCE TO:

André Junqueira Zaharenko, Rua Capitão João Carlos, 36, São Paulo, SP, CEP: 02926060, Brasil, Email: a.j.zaharenko@ig.com.br