Table 2 shows the peptide fragments predicted by Orbitrap-MS and the toxins that share these peptides. In addition, fourteen of these twenty nine sequences (in bold, Table 2) were predicted in digested fragments of both protein bands, eight were predicted only in the 71 kDa (underlined,
Table 2) and seven were predicted only in 150 kDa band. There was no phospholipase A2 activity in either crude venom or purified Sp-CTx as shown by the absence of hydrolysis halos at the concentrations tested (data not shown). To test whether the hemolytic activity of Sp-CTx is induced by pore formation in the cell membrane and to examine the role of colloid-osmotic shock in the Sp-CTx-induced hemolysis, saccharose and PEG of different molecular sizes were used. We compared the patterns of protection afforded by these EGFR inhibitor molecules up to 120-min (Fig. 3A); however, there were no changes in the hemolysis intensity after 8 min (data not shown). Saccharose and PEG 1000 (data not shown) were unable to abolish cell lysis and conferred an insignificant protection against hemolysis when compared to others PEG. PEG 1450 did not avoid the hemolytic effect induced by Sp-CTx, but it increased the time necessary to reach 50% of hemolysis (t1/2) from 2 min to 4 min approximately ( Fig. 3A). PEG 3350 increased t1/2 by 80% and the absorbance did not reach zero even after 120 min of Sp-CTx incubation (data not shown). Thus, only PEG 8000 was capable of giving
full protection selleckchem against hemolysis ( Fig. 3B). In the present study we demonstrated for the first time the pore-forming activity of Sp-CTx, a cytolytic toxin from the S. plumieri venom. This is also the first time that predicted sequences from Sp-CTx tryptic fragments are shown, which are shared by other fish venoms toxins. To achieve such results, we first optimized the purification method of Sp-CTx in order to improve the Sitaxentan protein yield and activity. Our group ( Andrich et al., 2010) had already purified Sp-CTx, a cytolytic toxin from S. plumieri fish venom, employing four chromatographic steps (two gel filtration and two anion exchange chromatographies). In the present study we
developed a new methodology to purify Sp-CTx with higher yield, through a reduced number of steps (saline precipitation followed by two chromatographic steps). This new method is time saving once it reduced the time to get the pure toxin from seven to only one day. Due to the lability of Sp-CTx, this time reduction was essential for the success of its purification. The time reduction also minimizes proteolytic nicking/hydrolysis of venom proteins by the endogenous proteases present in S. plumieri venom ( Carrijo et al., 2005). In addition, this new method improved the final yield to 13% (13 fold increase compared to the previous one) ( Andrich et al., 2010). The obtainment of higher amounts of Sp-CTx allowed us to further investigate its chemical and hemolytic properties.