Molecular and biochemical characterization of a cytolysin from the Scorpaena plumieri (scorpionfish) venom: evidence of pore formation on erythrocyte cell membrane.
Previously, a potent hemolytic toxin (Sp-CTx - 121 kDa) was isolated from Atlantic Scorpionfish Scorpaena plumieri venom. In the present work, we aimed to elucidate the action mechanisms involved in the hemolytic activity induced by this toxin, but to achieve our goal we faced the need to optimize its purification procedure in order to improve its activity and protein recovery. In this new method, Sp-CTx was purified to homogeneity through a combination of sequential ammonium sulfate precipitation and two chromatographic steps: hydrophobic interaction (Butyl HP) and anion exchange (Synchropak SAX 300). Orbitrap mass spectrometry analysis revealed that the amino acids sequences determined to Sp-CTx peptides are shared by other hemolytic toxins from fish venoms. The hemolytic activity of Sp-CTx upon rabbit erythrocytes was attenuated in the presence of osmotic protectants (polyethylene glycol polymers), and molecules larger than 6 nm in diameter inhibited cell lysis. This result strongly suggests that Sp-CTx may be a pore-forming protein, since it lacks phospholipase A2 activity. All these results contribute to the better understanding of Sp-CTx molecular/cellular actions in envenomation caused by S. plumieri. The results are also in agreement with previous reports of structural and functional similarities among piscine hemolytic toxins.
Programa de Pós-Graduação em Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES, Brazil.
Helena L
HL
Gomes
Filipe
F
Andrich
Consuelo L
CL
Fortes-Dias
Jonas
J
Perales
André
A
Teixeira-Ferreira
Dalton V
DV
Vassallo
Jader S
JS
Cruz
Suely G
SG
Figueiredo
D000818:Animals; D055598:Chemical Phenomena; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000187:drug effects*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects*; D005397:Fish Venoms / Q000737:chemistry*; D006461:Hemolysis / Q000187:drug effects; D010473:Perciformes; D054353:Perforin / Q000737:chemistry* / Q000302:isolation & purification; D054467:Phospholipases A2 / Q000378:metabolism; D011817:Rabbits
D005397:Fish Venoms; D054353:Perforin; D054467:Phospholipases A2
Fish venom; Hemolysis; Scorpaena plumieri; Scorpionfish; Sp-CTx