The first report on isolation of a new highly hemolytic toxin, Scatotoxin, from Scatophagus argus venomous bonny spines

Scatophagus argus of family Scatophagidae is a venomous fish. Rough spines insulated from scat possessed potent venom composed of several proteins. Envenomation is associated with local necrosis and severe pain. Following our previous report regarding the hemolytic activity of scat crude venom, this work aimed at purification and evaluation of its hemolytic protein, hereafter designated as Scatotoxin. Specimens were collected from coastal waters of the Persian Gulf, Iran. Proteins were extracted from bone tissue by solubilization buffers and subsequently refolded in a refolding buffer. Purification was performed by reverse-phase HPLC method using a linear gradient protocol. To evaluate the hemolytic activity of Scatotoxin, a quantitative microscopic assay was developed using cell counting by which measurement of activity of the least amount of a sample was achievable. Scatotoxin was isolated in 85% acetonitrile. It is an interesting highly hydrophobic protein. Because hemolysis was observed immediately, scatotoxin is considered a very fast-acting hemolytic agent. Scatotoxin indicated as a 72 kDa protein by SDS-PAGE. The amount of 0.5µg crude venom produced 100% hemolysis and HD50 determined at 0.18µg. HD50 for scatotoxin recorded at 0.003µg. High efficiency of both extraction method and microscopic-scale assay led to the reduction of collected specimens and consequently avoiding harmful effects on the Persian Gulf ecosystem. This issue is ethically important due to decreasing the number of samples too. Among the previously reported hemolytic proteins, Scatotoxin is the first report of a highly hydrophobic protein.