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Toxicon 60 (2012) 934–942

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Toxicon

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General biochemical and immunological characteristics of the venom from Peruvian Hadruroides lunatus

F. Costal-Oliveira a, C.G. Duarte a, R.A. Machado de Avila a, M.M. Melo c, K.C.F. Bordon d, E.C. Arantes d, N.C. Paredes e, B. Tintaya e, C. Bonilla e, R.E. Bonilla a, W.S. Suarez e, A. Yarleque f, J.M. Fernandez f, E. Kalapothakis b, Carlos Chávez-Olórtegui a,*

a Departamentos de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil b Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil c Escola de Veterinaria, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil d Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto-SP, Brazil e Instituto Nacional de Salud, Lima, Peru f Universidad Nacional Mayor de San Marcos, Lima, Peru

article info abstract

Article history: This communication describes the general biochemical properties and some immuno- Received 3 May 2012 logical characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which Received in revised form 1 June 2012 is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic Accepted 20 June 2012 to mice, the LD determined was 0.1 mg/kg and 21.55 mg/kg when the venom was Available online 29 June 2012 50 injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance Keywords: liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two Hadruroides lunatus scorpion venom Phospholipasic activity peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were LD50 Anti-H. lunatus sera produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World- serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector. Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license.

1. Introduction geographical distribution. This biodiversity has not, however, been properly studied. Peru is a country in western South America. It is Hadruroides (Pocock, 1893) is a scorpion genus included bordered on the north by Ecuador and Colombia, on the in the family Iuridae, subfamily Charaboctoninae. This east by Brazil, on the southeast by Bolivia, on the south by genus comprises sixteen species and there members Chile, and on the west by the Pacific Ocean. This nation has appear brown in color with darker stains and have median a rich and diverse herpetic and fauna, with wide size of 80 mm (Ochoa and Prendini, 2010; Maury, 1975). Hadruroides scorpions have been reported in Bolivia, Chile, Colombia, Ecuador, Peru, and Venezuela (Mello-Leitão, 1945; Esquivel de Verde, 1968; Kinzelbach, 1973; Maury, * Corresponding author. Departamento de Bioquímica e Imunologia, 1975; Cekalovic, 1983; Sissom and Fet, 2000), but are Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. CP: 486. CEP: 31270-901. Tel.: þ55 31 3409 2625; actually restricted to Ecuador, Peru, northern Chile, and fax: þ55 313 441 5963. several offshore islands, including the Galápagos E-mail address: [email protected] (C. Chávez-Olórtegui). (Cekalovic, 1966; Maury, 1975; Francke and Soleglad, 1981).

0041-0101 Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.toxicon.2012.06.013 F. Costal-Oliveira et al. / Toxicon 60 (2012) 934–942 935

Species of Hadruroides inhabit inter-Andean valleys, Pacific were fed weekly with cockroaches. The venom from desert, and dry forest habitats (Ochoa and Prendini, 2010). mature scorpions was obtained by electrical stimulation Hadruroides lunatus (“escorpion de los pedregales”)is (12 V) of the telsons. The venom collected in micropipettes the most medically relevant species in Peru. According to was diluted in ultrapure water, pooled and stored at 20 C the Health Ministry of Peru (Ministerio de Salud del Perú, until use. The protein concentration was determined by the 2004), the number of human envenomation cases repor- method of Lowry et al. (1951). ted has increased during recent years, with most incidents mature scorpions were collected in the occurring in the Central Coast of the country, which region of Belo Horizonte and maintained at the “Seção de corresponds with the main area of geographical distribu- Animais Peçonhentos” of Ezequiel Dias Foundation tion of H. lunatus scorpions (Zavaleta et al., 1981). Severe (FUNED) of Belo Horizonte, Brazil. The crude venoms were toxic effects by H. lunatus stings have not been noted in obtained by electrical stimulation of the telsons, lyophi- humans; however, intense pain, edema and ulceration are lized and stored at 20 C in the dark until use. The venoms frequently described as symptoms (Zavaleta et al., 1981). from the scorpions Androctonus australis hector and Cen- Different approaches are adopted for the treatment of truroides sculpturatus were obtained from the Laboratoire scorpion envenomations such as local care, analgesics and de Biochimie, Faculté de Médecine, Marseille, France and antihistaminics (Ministério de Salúd, Peru, 2004). Never- from Sigma Chemical Company, St. Louis, USA, respectively. theless, there are no scientific data to support these treat- Male and female Swiss and C57BL/6 mice (weighing 18– ments. The Instituto Nacional de Salud (INS) in Lima, Peru 22 g) and male Wistar rats (weighing 110–150 g) were does not produce specific scorpion anti-venon (Ministério maintained at the Centro de Bioterismo of the Instituto de de Salúd, Peru, 2004). Consequently, the treatment of Ciências Biológicas of the Universidade Federal de Minas scorpion envenomations with specific anti-venom for Gerais (UFMG), Belo Horizonte, MG, Brazil. All Peruvian species does not exist. received water and food under controlled environmental Very little is known about the structural and functional conditions. The experimental protocols were approved by characteristics of Peruvian scorpion venoms. The first the “Ethics Committee on the Use of Laboratory Animals of toxicological information was obtained from research on UFMG” (CETEA-UFMG). Eight- to nine-week-old New Zea- the H. lunatus species (Delgado and Pesce, 1967; Aguilar, land rabbits were used to produce anti-H. lunatus and anti- 1968; Aguilar and Meneses, 1970 and Zavaleta et al., T. serrulatus sera. Animals were maintained and handled as 1981). The pharmacological effects described by Zavaleta described previously. et al. (1981) showed that H. lunatus crude venom has a low lethality in mice (LD50, 68 mg/kg i.p.) and, in dogs, 2.2. Toxic activities of H. lunatus venom induces a fall in blood pressure. Neurotoxic activity in insects, crustaceans and mice and antibacterial peptides 2.2.1. Determination of median lethal dose (LD50) from the Hadruroides sp. crude venoms were showed by The lethality was assessed by intraperitoneal (i.p.) and Escobar et al. (2002) and Escobar and Flores, (2008). intracranial (i.c.) routes. For the intraperitoneal route, However, proteolytic and phospholipasic activities of this groups of four mice were injected with different doses of scorpion venoms are not shown in these studies. Despite venom (from 11.53 mg to 32.95 mg per kg of body weight) these published works, the studies with Peruvian scorpions dissolved in 0.5 mL of PBS–BSA 0.1%. For the intra- are still very preliminary. cerebroventricular route, groups of six mice were injected In view of the lack of information on the general with various doses of venom (from 0.075 mg to 0.8 mg per characteristics of Hadruroides scorpion venoms, the main kg) dissolved in 5 mL of PBS–BSA 0.1%. Twenty four hours goal of this work was to report additional biochemical later deaths were recorded and the LD50 was then calcu- and toxic characterization of H. lunatus scorpion venom. lated by Probit analysis (Finney, 1971). In this paper, the hyaluronidase, proteolytic, phospholi- pase, cardiotoxic and lethal activities of H. lunatus crude 2.2.2. Determination of proteolytic activity (PA) venom were investigated. This communication also Proteolytic activity was measured with dimethylcasein describes the separation of the soluble venom compo- (Sigma) as described by Lin et al. (1969) with modifications nents by SDS-PAGE and by high performance liquid described in Sanchez et al. (2000). Dilutions corresponding chromatography (HPLC). Furthermore, the last part of this to 5, 10, 20 and 40 mg of venom were used and absorbance study shows, some immunological characteristics of values were determined at 340 nm. One unit was defined as soluble whole venom using specific polyclonal rabbit anti- DA 340 nm/min. Activity was expressed relative to protein H. lunatus antibodies. concentration (mg).

2. Materials and methods 2.2.3. Determination of phospholipase A2 (PLA2) activity PLA2 activity was measured using an indirect hemolytic 2.1. Animals and venoms assay (Gutierrez et al., 1988). Increasing concentrations of H. lunatus crude venom (0.0625, 0.125, 0.25, 0.5 and 1 mg) were H. lunatus scorpions were collected in the region of prepared in a final volume of 15 mL in PBS and added to 2 mm Atocongo (Lima, Peru) and maintained in the herpetarium wells in agarose gels (0.8% in phosphate buffered saline, pH of the Centro Nacional de Producción de Biologicos of 8.1) containing 1.2% sheep erythrocytes, 1.2% egg yolk as Instituto Nacional de Salud (INS), in Lima, Peru. Scorpions a source of lecithin, and 100 mM CaCl2. The main fractions were maintained in plastic boxes with water ad libitum and obtained in the purification of the venom by HPLC were also 936 F. Costal-Oliveira et al. / Toxicon 60 (2012) 934–942 tested. Plates were incubated at 37 C for 18 h and the 2.3. Chromatography and mass spectrometry assays diameters of the hemolytic halos were measured. As a control, 15 mL of PBS was tested. One unit (Minimum Chromatographic fractionation of H. lunatus venom was Phospholipasic Dose – MPD) corresponds to a minor performed using high performance liquid chromatography concentration of venom which produced a hemolytic halo (HPLC). Briefly, 1 mg of crude venom was applied to of 1 cm diameter. Experiments were conducted in duplicate. a reverse phase column. The column used in this assay was a Shimadzu-Pack CLC-ODS C18 (6 150 mm) eluted at 2.2.4. Determination of hyaluronidase activity (HA) 1 mL/min with a linear gradient of 0.1% TFA in water and Hyaluronidase activity of the venom and its molecular acetonitrile, solutions A and B, respectively. After column mass determination was analyzed by sodium dodecyl equilibration the venom fractions were separated with sulfate-polyacrylamide gel electrophoresis and performed a linear gradient from solution A to 60% solution B, running according to Cevallos et al. (1992).Briefly, SDS-PAGE gels for 60 min. were prepared with hyaluronan, which was incorporated Fractions were then subjected to MALDI-TOF-TOF anal- into the gels as a hyaluronidase substrate in the 10% yses. MS analysis was performed using a MALDI-TOF-TOF resolving gel at a final concentration of 0.5 mg/mL. AutoFlex IIIÔ (Bruker Daltonics) instrument in positive/ Venom samples (10 and 5 mg), dispersed in Laemmli reflector mode controlled by the FlexControlÔ software. buffer under non-reducing conditions and room temper- Instrument calibration was achieved by using Peptide ature, were electrophoresed at 90 V at room temperature Calibration Standard IV (Bruker Daltonics) as a reference until the indicator reached the end of the gel. After and using sinapinic acid as a matrix. The peak was spotted electrophoresis, the gel was washed twice in 5% Triton X- to MTP AnchorChipÔ 400/384 (Bruker Daltonics) targets 100 in sodium phosphate buffer 0.1 M, pH 5.8, with using standard protocols for the dried droplet method. 0.15 M NaCl for 1 h, once in 0.05% Triton X-100 in buffer pH 5.8 for an hour and finally in buffer pH 5.8 without 2.4. Immunological studies Triton X-100 for 10 min. All these steps were performed at room temperature. Subsequently, the gel was placed in 2.4.1. Immunization protocols buffer without Triton X-100 and incubated at 37 C for the Adult New Zealand female rabbits were used for the desired amount of time. After incubation, the gels were production of anti-H. lunatus and anti-T. serrulatus venom washed twice for 15 min in 0.015 M Tris–HCl, pH 7.95 and antibodies. After collection of pre-immune sera, the then stained under gentle rotation for at least 5 h in the animals received an initial subcutaneous injection of 100 mg dark. A stock solution of 0.1% Stains-all (1-ethyl-2-[3-(1- of whole venom in complete Freund’s adjuvant (day 1). ethylnaphthol [1,2-d] thiazolin-2-ylidene)-2- Three booster injections were made subcutaneously 14, 21 methylpropenyl] Naphthol [1,2-d] thiazolium bromide; and 28 days later with a lower dose (50 mg) in incomplete Cat. No. 2718 fromEastman Kodak Company, Rochester, Freund’s adjuvant. The animals were bled one week after NY, USA) in pure formamide was stored in a dark the last injection. container. The dye solution was freshly prepared by combining 5 mL dye stock with 5 mL of formamide, 20 mL 2.4.2. Indirect ELISA and immunoblotting assays of isopropanol, 1.5 mL of 1 M Tris–HCl, pH 7.95, and Maxisorp microtitration plates (Nalge Nunc, USA) were deionized water to a volume of 100 mL (Green et al., coated overnight at 5 C with 100 mLofa10mg/mL solution 1973). The gel was destained in 5% formamide and 20% of H. lunatus, T. serrulatus, A. australis or C. sculpturatus isopropanol in 0.015 M Tris–HCl, pH 7.95, until bands of whole venom in carbonate buffer pH 9.6. After blocking (3% activity become clear. The protein molar mass standards powdered milk in PBS) and washing (0.05% Tween-saline), were always separated at the extreme end of the gel plate sera from pre-immune and immune rabbit were added in and following electrophoresis, the line was carefully different dilutions and incubated for 1 h at 37 C. Plates sectioned and stained with Coomassie brilliant blue were washed and incubated with anti-rabbit IgG conju- R-250. gated with peroxidase diluted 1:4000, for 1 h at 37 C. After washing, 100 mL of o-phenylenediamine (0.33 mg/mL in 2.2.5. Determination of cardiotoxic activity citrate buffer, pH 5.2, in the presence of 0.04% hydrogen CK and CK–MB levels in the serum of envenomed rats peroxide) was added to the wells. The reaction was stopped were determined as a measured of the cardiotoxicity of H. after 20 min by the addition of 20 mL of a 1:20 sulfuric acid lunatus venom. Groups of six Wistar rats were injected solution. Absorbance values were determined at 490 nm intraperitoneally (i.p.) with 750 mg of soluble venom or using an ELISA plate reader (BIO-RAD, 680 models). ultra-pure water (control). The animals were kept under Duplicate readings were taken for all samples and the inhalation anesthesia with morphine (2.5 mg/kg) and means were calculated. diazepam (2.5 mg/kg), injected via the intramuscular route For the immunoblotting, an SDS-PAGE gel using H. (Flecknell et al., 1996). After 30 min of envenoming, blood lunatus venom was run according to the method of was collected by cardiac puncture. Blood was then centri- Laemmli (1970) using 12.5% gels and transferred onto fuged (3000 rpm for 5 min) and serum used for biochem- nitrocellulose membrane (Towbin et al., 1979). The ical analysis. The levels of creatine kinase isoenzyme MB membrane was blocked with PBS-Tween 0.3% for 1 h. After (CK–MB) and total creatine kinase (CK) were measured washing three times for 5 min with PBS-Tween 0.05%, the using commercial kits from Bioclin (Quibasa, Brazil) and membrane was incubated with anti-H. lunatus rabbit serum a Thermo Plate Analyzer Basic instrument. (1:1500) for 1 h and 30 min. The membrane was washed F. Costal-Oliveira et al. / Toxicon 60 (2012) 934–942 937

(PBS-Tween 0.05%) more three times and immunoreactive Q), pooled and stored at 20 C until use and never proteins were detected using anti-rabbit IgG conjugated to lyophilized. peroxidase (1:8000) for 1 h at room temperature. After The proteolytic activity (caseinase) of H. lunatus venom washing three times for 5 min with PBS-Tween 0.05%, blots was detected, for the first time, by the dimethylcasein were developed using DAB/chloronaphthol according to method (Lin et al., 1969; Sanchez et al., 2000). The manufacturer’s instructions. proteolytic activity of the venom was determined as 0.46 U/ mg min and showed a dose dependent pattern (Fig. 1A). 3. Results and discussion This activity has already been investigated for the H. lunatus venom, by Escobar and co-workers in 2002. However, in 3.1. General venom characterization this study, no proteolytic activity was found. This difference may be due to the method used in the previous work that The lethality of the H. lunatus soluble venom to may not have a comparable sensitivity to the method mammals was examined using mice. After intracranial (i.c.) chosen in this study. Enzymes with gelatinolytic activity and intraperitoneal injection (i.p.) toxic and lethal effects were detected in T. bahiensis and T. serrulatus venom were observed. The LD50 value was determined as 0.1 mg/ (Almeida et al., 2002). For these species Magalhães (1946) kg and 21.55 mg/kg (respectively). Injected mice displayed suggested that proteolytic enzymes were present based typical symptoms of intoxication such as excitability, on observations of necrosis, hemolysis and gangrene agitation, salivation, eye secretions, sweating, convulsions following experimental injection in different animals. and paralysis of legs. The symptoms lasted for 30–120 min Necrosis is reported as one of the symptoms in humans before death. The observed symptoms closely resemble stung by H. lunatus scorpions (Anales de la Facultad de those produced by the venom of scorpions of the Medicina, 1967). The role for proteolytic enzymes in scor- genera Centruroides or Tityus (Possani et al., 1977). The pion venoms is not very clear, but they may be related to venoms from some species of Brazilian scorpions were tissue permeability, acting as spreading factors for the analyzed regarding their lethality in mice by Nishikawa and venom. These enzymes may also have a direct toxic effect in co-workers, in 1994 via intraperitoneal injection, the same the development of pancreatitis presented by some enve- used by us. In this study, the venoms were grouped as nomed patients (Almeida et al., 2002; Renner et al., 1983). highly toxic {Tityus stigmurus (LD50 ¼ 0.773 mg/kg), Tityus The H. lunatus venom also displays phospholipase and bahiensis (LD50 ¼ 1.062 mg/kg) and T. serrulatus hyaluronidase activities (Figs. 1B and 2). The phospholipase (LD50 ¼ 1.160 mg/kg)}; moderately toxic {Tityus cambridgei activity was examined through an indirect hemolytic assay, (LD50 ¼ 12.136 mg/kg)} and practically non-toxic {Rhopa- as described by Gutierrez in 1988 and is dose dependent. lurus agamemnon (LD50 ¼ 36.363 mg/kg) and Brotheas The minimum phospholipasic dose (MPD), which is the fi amazonicus (LD50 ¼ 90.909 mg/kg)}. In view of the results dose capable of producing a 1 cm halo, was de ned as observed in our experiments and compared to the previous 0.125 mg of soluble venom. Phospholipase activity has been data, H. lunatus venom can be classified as moderately described for some scorpion venoms (Schwartz et al., fi toxic. The value found for LD50 of the venom of H. lunatus 2008), and phaiodactylipin was the rst molecule from (i.p. route) was more than three times lower than that the venom of a scorpion belonging to the Iuridae family to found by Zavaleta et al. (1981). This divergence can be be described (Valdez-Cruz et al., 2007). This scorpion explained by the fact that in our experiments the venom phospholipase belongs to the A2 family, class III. It is collected was immediately diluted in ultrapure water (milli a heterodimeric protein with neurotoxic activity that acts

Fig. 1. Proteolytic and PLA2 activities of Hadruroides lunatus venom. The proteolytic activity (Fig. 1A) was determined using dimethylcasein as a substrate. The absorbance value (at 340 nm) showed the proteolytic activity of 5, 10, 20 and 40 mg of crude venom. One unit was defined as DA 340 nm/min, and the activity was expressed relative to protein concentration (mg). PLA2 activity (Fig. 1B) was determined via the indirect hemolytic assay in agarose gel containing rabbit erythrocytes, egg yolk and CaCl2. The samples (15 mL) containing different concentrations of H. lunatus venom were added to 2 mm wells on agarose, incubated at 37 C for 18 h and the diameters of the hemolytic halos measured. 938 F. Costal-Oliveira et al. / Toxicon 60 (2012) 934–942

corresponding to 40 kDa as being responsible for the hyaluronidase activity of H. lunatus venom. The presence of hyaluronidase in scorpion venoms is probably related to the spread and absorption of venom’s toxic components and may affect the stability of blood vessel walls (Veiga et al., 2001; Seyedian et al., 2010). In addition, it was recently shown that hyaluronidase combined with phos- pholipase and citolytic peptides from insect venoms can promote an IgE or IgG allergen antibody response in mice or in susceptible humans (King and Wittkowski, 2011). 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 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 Fig. 2. Determination of hyaluronidase activity of Hadruroides lunatus suggest in this study that H. lunatus venom has cardiotoxic venom using zymogram. Hyaluronidase activity was analyzed by SDS–PAGE effects, possibly through the action of neurotoxins acting on on a 5% (w/v) concentration and 10% separation acrylamide gel co- voltage gated ion channels present in the heart (Chen and polymerized with hyaluronic acid as described in the Material and Heinemann, 2001; Korolkova et al., 2004). methods. Lanes 2 and 3 contain 10 and 5 mg of crude H. lunatus venom. The molecular weight marker of 45 kDa is show in lane 1. 3.2. Venom fractionation by inhibiting the ryanodine receptor (Zamudio et al., 1997). The soluble venom of H. lunatus was fractionated by Besides their importance in digestion, venom phospholi- HPLC and showed more than 20 components (Fig. 4A). As pases may also be related to other toxic symptoms such as with other chromatographic profiles of scorpion venoms inflammation, platelet aggregation, mionecrosis, hemo- (Batista et al., 2004), the separation in C18 reverse column lysis, neurotoxicity and cardiotoxicity (Lambeau and is completed at approximately 60 min of the gradient, at Lazdunski, 1999). The hyaluronidase activity appears a flow rate of 1 mL/min. According to the authors ubiquitous in all venom samples obtained from scorpion mentioned above, the fractionated components during the venoms (Pessini et al., 2001; Seyedian et al., 2010). first 20–40 min of the gradient would be the minor þ þ Using the zymogram technique (Cevallos et al., 1992), we peptides corresponding to K - and Na - channel neuro- have identified a component with a molecular mass toxins. It is known that most scorpion toxic peptides have

Fig. 3. Serum levels of total creatine kinase (CK) and creatine kinase isoenzyme MB (CK–MB) in rats envenomed with Hadruroides lunatus venom. Animals were injected with 750 mg of soluble venom. Control rats were injected with PBS alone. (A) CK serum levels in rats of the control group and of the venom group. (B) CK– MB serum levels of the control animals and of the envenomed group (*, p < 0.05 vs. control, values shown represent mean plus standard errors). F. Costal-Oliveira et al. / Toxicon 60 (2012) 934–942 939

Fig. 4. General analysis of Hadruroides lunatus venom fractions. For HPLC separation 1 mg of crude venom was applied to a reverse phase column (Shimadzu-Pack CLC-ODS C18) (Fig. 4A). After column equilibration the venom fractions were separated with a linear gradient from solution A to 60% solution B (dashed line), running for 60 min. The chromatographic profile is shown by the black line. Phospholipase activity (PLA2) of some fractions was performed using an indirect hemolytic assay as previously described. The fractions 10, 11, 12, 14, 15, 16, 17, 18 and 19 presented phospholipase activity. The size of the halo produced by each fraction is shown by the gray line located above the respective fraction. Fig. 4B shows the spectrophotometric analysis of the major phospholipasic fraction from H. lunatus venom (fraction15). Two molecular masses were identified by MALDI-TOF-TOF analysis, one of 11914.5 and another of 13650.6 Da.

molecular masses lower than 10 kDa. These basic peptides potential and cross-reactivity of rabbit anti-H. lunatus are neurotoxins of low molecular mass that bind to ion serum. Immune sera anti-H. lunatus and anti-T. serrulatus channels with high affinity, exerting their noxious effect (for comparative purposes), were raised in rabbits and their (Catterall et al., 2007). These small proteins may be reactivities against H. lunatus, T. serrulatus, C. sculpturatus responsible for the typical symptoms of neurotoxic enve- and Androctonus australis hector venoms evaluated. Fig. 5 noming observed in inoculated mice. Several components shows the ELISA (absorbance at 490 nm) at different purified after RT (retention time) 30 min showed PLA2 serum dilutions (1:100 to 1:12,800). Each specific serum activities (peaks 10 to 19, except the fraction 13). Fraction reacted strongly against their own venom antigens. 15 (from 35 to 40 min RT), which showed highest PLA2 Notably, sera against Peruvian venom (Fig. 5A) reacted activity, was further analyzed by MALDI–TOF and the moderately with their Brazilian and North American individual components clearly identified. The molar masses counterparts and poorly with the venom from A. australis (Fig. 4B) found were 11,914.5 Da and 13,650.6 Da. (North African). The serum produced against T. serrulatus (Fig. 5B) recognized strongly the Peruvian and North 3.3. Immunological studies American venoms coated on the ELISA plates and reacted moderately with the North African scorpion venom. The In the final part of our study and as a preliminary step in above observations suggest the presence of antigenic the production of an anti-H. lunatus anti-venom with identities or common epitopes across the four scorpion therapeutic properties, we have attempted to study by genera studied. However, the recognition of anti-H. lunatus ELISA and immunoblotting the antigenic/immunogenic and anti-T. serrulatus venom antibodies against the venoms 940 F. Costal-Oliveira et al. / Toxicon 60 (2012) 934–942

Fig. 5. ELISA cross reactivity of anti-H. lunatus and anti-T. serrulatus rabbit sera antibodies against different scorpion venoms. The reactivity of sera (dilutions 1:100 to 1:12800) from rabbits immunized with H. lunatus (Fig. 5A) and T. serrulatus (Fig. 5B) toward venoms (10 mg/mL) from the H. lunatus, T. serrulatus, C. sculpturatus and A. a. hector was tested. Values are means SEM of triplicates. The absorbance of pre-immune serum used as negative control was subtracted. of the American regions was significantly higher than responsible for the cross-reactivity, observed in the ELISA against African scorpion venom. The antigen–antibody among the four scorpion venoms analyzed in this study. reactivity was also examined using Western blotting with Although the efficiency of immunotherapy in treating H. lunatus venom and rabbit polyclonal anti-H. lunatus anti- patients stung by scorpion remains controversial venom. From the study (Fig. 6), at least six antigenic (Theakston et al., 2003), certainly a complete analysis of the components were identified. Bands were observed around neutralizing potency of rabbit anti-H. lunatus anti-venom 7,15, 45 and 66 kDa. Molar masses higher than 66 kDa were can provide convincing support that the anti-venom could also recognized by anti-H. lunatus antibodies. The presence be an effective means of neutralizing the toxic effects of of 45 kDa bands in H. lunatus venom observed by Western H. lunatus scorpion toxins. Previous studies in our group in blotting is due to the presence of the enzyme hyaluroni- Brazil (de Resende et al., 1995), strongly support the use of dase. Hyaluronidase, is present in all venom samples anti-venom therapy in scorpionism, but the success of this obtained from scorpions (Pessini et al., 2001;;Seyedian therapy remains dependent of the quality of the anti- et al., 2010). Antibodies against hyaluronidase can be venoms and the spread at which the treatment is provided. F. Costal-Oliveira et al. / Toxicon 60 (2012) 934–942 941

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