Total serum creatine

kinase (CK) and its isoenzyme MB (CK–MB) are well established and widely accepted markers for the diagnosis and follow-up of heart injury or myocardial infarction (Bachmaier et al., 1995). Biochemical analyses showed an increase in CK and CK-MB levels (Fig. 3A and B, respectively). Increased CK concentrations were observed see more in envenomed animals (4850 UI/mL) compared to the control group (injection of PBS) (1293 UI/mL). Levels of CK–MB were also higher in the envenomed group (1980 UI/mL) than in the control group (413 UI/mL). We have demonstrated previously, in dogs envenomed with Tityus fasciolatus scorpion venom ( Pinto et al., 2010), that the occurrence of myocardium damage is correlated with high serum levels of CK and CK–MB. As we

also observed higher levels of these two markers of heart injury of the envenomed rats, we suggest in this study that H. lunatus venom has cardiotoxic effects, possibly through the action of neurotoxins acting on voltage gated ion channels present in the heart ( Chen and Heinemann, 2001; Korolkova et al., 2004). The soluble venom of H. lunatus was fractionated by HPLC and showed more than 20 components ( Fig. 4A). As with other chromatographic profiles of scorpion venoms ( Batista et al., 2004), the separation in C18 reverse column is completed at approximately 60 min of the Natural Product Library cell line gradient, at a flow rate of 1 mL/min. According to the authors mentioned above, the fractionated components during

the first 20–40 min of the gradient would be the minor peptides corresponding to K+- and Na+- channel neurotoxins. It is known that most scorpion Phloretin toxic peptides have molecular masses lower than 10 kDa. These basic peptides are neurotoxins of low molecular mass that bind to ion channels with high affinity, exerting their noxious effect ( Catterall et al., 2007). These small proteins may be responsible for the typical symptoms of neurotoxic envenoming observed in inoculated mice. Several components purified after RT (retention time) 30 min showed PLA2 activities (peaks 10 to 19, except the fraction 13). Fraction 15 (from 35 to 40 min RT), which showed highest PLA2 activity, was further analyzed by MALDI–TOF and the individual components clearly identified. The molar masses ( Fig. 4B) found were 11,914.5 Da and 13,650.6 Da. In the final part of our study and as a preliminary step in the production of an anti-H. lunatus anti-venom with therapeutic properties, we have attempted to study by ELISA and immunoblotting the antigenic/immunogenic potential and cross-reactivity of rabbit anti-H. lunatus serum. Immune sera anti-H. lunatus and anti-T. serrulatus (for comparative purposes), were raised in rabbits and their reactivities against H. lunatus, T. serrulatus, C. sculpturatus and Androctonus australis hector venoms evaluated. Fig. 5 shows the ELISA (absorbance at 490 nm) at different serum dilutions (1:100 to 1:12,800).