Identification of hemolytic and neuroactive fractions in the venom of the sea anemone Bunodosoma cangicum

Abstract

Sea anemones are a rich source of biologically active substances. In crayfish muscle fibers, Bunodosoma cangicum whole venom selectively blocks the I K(Ca) currents. In the present study, we report for the first time powerful hemolytic and neuroactive effects present in two different fractions obtained by gel-filtration chromatography from whole venom of B. cangicum. A cytolytic fraction (Bcg-2) with components of molecular mass ranging from 8 to 18 kDa elicited hemolysis of mouse erythrocytes with an EC 50 = 14 µg/ml and a maximum dose of 22 µg/ml. The effects of the neuroactive fraction, Bcg-3 (2 to 5 kDa), were studied on isolated crab nerves. This fraction prolonged the compound action potentials by increasing their duration and rise time in a dose-dependent manner. This effect was evident after the washout of the preparation, suggesting the existence of a reversible substance that was initially masking the effects of an irreversible one. In order to elucidate the target of Bcg-3 action, the fraction was applied to a tetraethylammonium-pretreated preparation. An additional increase in action potential duration was observed, suggesting a blockade of a different population of K + channels or of tetraethylammonium-insensitive channels. Also, tetrodotoxin could not block the action potentials in a Bcg-3-pretreated preparation, suggesting a possible interaction of Bcg-3 with Na + channels. The present data suggest that B. cangicum venom contains at least two bioactive fractions whose activity on cell membranes seems to differ from the I K(Ca) blockade described previously.

Avaliação da atividade antiinflamatória e analgésica do extrato bruto hidroalcoólico do zoantídeo Palythoa caribaeorum (Duchassaing & Michelotti, 1860)

“…No Brasil existem alguns antecedentes de estudos químicos em cnidários, principalmente anêmonas e octocorais. Freitas e colaboradores trabalham desde a década do 80 com substancias biactivas de anêmonas-do-mar, principalmente com Bunodosoma caissarum e B. cangicum, encontrando atividade antimitótica, hemolítica, neurotóxica e antitumoral em câncer de mama humano Elena, 1991;Lagos et al, 2001;Malpezzi et al, 1995;Oliveira et al, 2004).…”

Biochemical and Electrophysiological Characterization of Two Sea Anemone Type 1 Potassium Toxins from a Geographically Distant Population of Bunodosoma caissarum

“…When this toxic mixture is applied to a Sephadex G-50 gel-filtration column, the peptide content of the venom is separated from enzymes, such as phospholipases and cytolysin [26,27,28]. Gel filtration of B. caissarum venom on Sephadex G-50 yielded five fractions named Fraction I to V (FrI–FrV) (Figure 2A), as previously described for the venom of B. cangicum [26] and B. caissarum population from the southeastern coast of Brazil [29]. Gel-filtration Fraction III (FrIII) from B. caissarum SPSPA population had the highest neurotoxicity when tested on swimming crabs ( Callinectes danae ) (data not shown), and it was further purified by reverse-phase high performance liquid chromatography (rp-HPLC) (Figure 2B).…”

Combining multidimensional liquid chromatography and MALDI–TOF-MS for the fingerprint analysis of secreted peptides from the unexplored sea anemone species Phymanthus crucifer

“…a) Short molecular mass range covered by gel filtration Previous fingerprinting analyses of sea anemone secretions [17,21] did not exploit the whole peptidomic range, comprising 1-10 kDa fractions from gel filtration chromatography. Instead, a shorter range was collected, 2-5 kDa, defined as the neurotoxic fraction [27]. However several classes of biologically peptides from sea anemones fall outside that mass range, such as protease inhibitors, type 2 potassium channel toxins, cytolytic peptides and small crab-paralyzing toxin.…”

“…In our study we collected all fractions within the complete peptidomic range of 1-10 kDa to include peptides of interest with molecular masses outside the 2-5 kDa range previously defined as the neurotoxic fraction [27] and analyzed in peptidomic approaches [17,21] of sea anemones. Currently, the separation of the peptide pool from the sea anemone secretion has been done by gel filtration in Sephadex G-50.…”

“…Nonetheless, other gel filtration media (FPLC and HPLC) are suitable such as Superdex Peptide [67] and Superdex 30 [68][69][70], which provide faster separations, higher resolution and appropriate selectivity for fractionating the 1-10 kDa peptides. The column can be calibrated by using molecular weight standards as described by Lagos [27] (a similar procedure was used in the present work) or by SDS-PAGE analysis of eluting fractions [28]. B) The use of multidimensional approaches including ionexchange chromatography.…”