6 magalhães et al 2008 a hyaluronidase from potamotrygon motoro (freshwater

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6 magalhães et al 2008 a hyaluronidase from potamotrygon motoro (freshwater

  1. 1. Toxicon 51 (2008) 1060–1067 A hyaluronidase from Potamotrygon motoro (freshwater stingrays) venom: Isolation and characterization$ Marta R. Magalha˜ esa , Nelson Jorge da Silva Jr.a , Cirano J. Ulhoab,Ã a Centro de Estudos e Pesquisas Biolo´gicas, Departamento de Biologia, Universidade Cato´lica de Goia´s, 74.605-010, Goiaˆnia, GO, Brazil b Departamento de Cieˆncias Fisiolo´gicas (ICB), Universidade Federal de Goia´s, 74.001-940 Goiaˆnia, GO, Brazil Received 13 September 2007; received in revised form 21 December 2007; accepted 28 January 2008 Available online 2 February 2008 Abstract Freshwater stingrays (Potamotrygon motoro) are known to cause human accidents through a sting located in its tail. In the State of Goia´ s, this accident happens especially during the fishing season of the Araguaia River. The P. motoro venom extracted from the sting presented hyaluronidase activity. The enzyme was purified by gel filtration on Sephacryl S-100 and ion-exchange chromatography on SP-Sepharose. A typical procedure provided 376.4-fold purification with a 2.94% yield. The molecular weight of the purified enzyme was 79 kDa as estimated by gel filtration on Sephacryl S-100. The Km and Vmax values for hyaluronidase, using hyaluronic acid as substrate, were 4.91 mg/ml and 2.02 U/min, respectively. The pH optimum for the enzyme was pH 4.2 and maximum activity was obtained at 40 1C. The hyaluronidase from P. motoro was shown to be heat instable, being stabilized by bovine albumin and DTT, and inhibited by Fe2+ , Mn2+ , Cu2+ and heparin. r 2008 Elsevier Ltd. All rights reserved. Keywords: Freshwater stingrays; Potamotrygon motoro; Venom; Hyaluronidase; Properties 1. Introduction Stingrays are found around the world in tempe- rate and tropical seas. They are also found in Atlantic rivers of tropical and temperate South America, Equatorial Africa and, at least, one Indo- Chinese river system, the Mekong river of Laos (Caras, 1974). In spite of being not aggressive, from the point of view of public health, stingrays are the most significant venomous fish in the world (Junghanss and Bodio, 2006). These fishes have one or more stings at the base of their tails, which have serrated edges and a very sharp tip. Its position on the tail, certainly, is responsible for the effectiveness of the defensive response when it is stepped on its back or badly handled. In these cases, a powerful strike blow of the tail towards the stimulus causes the penetration of the sting into the body of the victim. The sting is covered by an epithelium that possesses great quantities of gland- ular cells which produce venom when compressed ARTICLE IN PRESS www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.01.008 $ Ethical statement: Cirano J. Ulhoa declares that all procedures used in this study are in accordance with the Brazilian College of Animal Experimentation (http://www.cobea.org.br). All animals used were free again in the Crixa´ s-Ac-u´ River, their natural habitat. ÃCorresponding author. Tel.: +55 62 35211494; fax: +55 62 35211190. E-mail addresses: ulhoa@icb.ufg.br, ulhoa@icb1.ufg.br (C.J. Ulhoa).
  2. 2. during the penetration, spreading their content into the tissues of the victim (Castex and Loza, 1964; Castex, 1965; Halstead, 1971). South American freshwater stingrays are included in the Potamotrygonidae family, which comprise three valid genera: Plesiotrygon, Paratrygon and Potamo- trygon, the last being more diversified, with 19 described species (Charvet-Almeida et al., 2002; Carvalho et al., 2003). In accidents provoked for freshwater stingrays, the victim complains of intense pain, relating it with burning. Around the wounded spot appears erythema and edema, characterizing the first phase of envenomation. Then it develops a central necrosis causing, in the affected area, tissue flabbiness and formation of a pale pink deep ulcer, well cut, which evolves slowly, being a peculiar characteristic of this kind of envenomation (Castex, 1965; Haddad et al., 2004; Cook et al., 2006; Clark et al., 2007). Few studies about the toxic activities of fresh- water stingrays venom have been developed. The lack of data is mainly due to the difficulty to extract venom, and it is very difficult and dangerous to capture the animals. The amount of venom is very low, and likewise it is thermolabile (Haddad et al., 2004). The first study about the biochemistry and pharmacology properties of stingrays venom was carried out by Russell and Van Harreveld (1954), which demonstrated cardiovascular effects of Ur- obatis helleri venom. Rodrigues (1972) isolated an active principle of freshwater stingray Potamotrygon motoro venom with cholinergic activity on ileum of guinea pigs and hypotensive activity when managed by intravenous injection in rats. Russell (1953) indicated the presence of polypeptides of high molecular mass, serotonin and enzymatic activity of phosphodiesterase and 50 -nucleotidase in marine stingray venom. Recently, we have detected 50 - nucleotidase, phospholipase, acid phosphatase, hyaluronidase, caseinolytic, gelatinolytic and elasti- nolytic activities in P. motoro venom obtained from animals of Crixa´ s-Ac-u´ River (Goia´ s, Brazil) (Ma- galha˜ es, 2001). Caseinolytic, gelatinolytic and hya- luronidase activities were identified in Potamotrygon falkneri venom (Haddad et al., 2004). In a comparative study of Potamotrygon scobina and Potamotrygon orbignyi venoms, Magalha˜ es et al. (2006) identified significant edematogenic and noci- ceptive response and necrosis in both venoms. Conceic-a˜ o et al. (2006) isolated a vasoconstrictor peptide from P. orbignyi venom with 1001.52 Da. Barbaro et al. (2007), comparing the extracts from the tissue of marine and freshwater stingrays Dasyatis guttata and P. falkneri, observed edemato- genic, gelatinolytic, caseinolytic and fibrinogenoly- tic activities in both extracts. Nociceptive activity was verified in both tissue extracts; however, P. falkneri presented a two-fold higher activity than D. guttata tissue extract. Lethal, dermonecrotic, myotoxic and hyaluronidase activities were ob- served only in the tissue extract of P. falkneri. Hyaluronidases (EC 3.2.1.35) are enzymes that naturally cleave hyaluronic acid, which is a major component of the extracellular matrix of vertebrates (Kreil, 1995). These enzymes are not toxic by themselves, but can enhance local systemic envenoma- tion by increasing the absorption and diffusion rates of the venom through the victim’s tissues since it catalyzes the hydrolysis of the glucosaminoglycans, this being called the spreading factor (Duran-Reynals, 1936). Hyaluronidase enzyme has been reported in venom of snakes, scorpions, bee, stonefish, lizards and spiders (Owen, 1983; Tu and Hendon, 1983; Poh et al., 1992; Kemparaju and Girish, 2006; Morey et al., 2006; Nagaraju et al., 2007). Recently, hyaluronidase activity was reported in the freshwater stingrays’ crude venom (Magalha˜ es, 2001; Haddad et al., 2004; Barbaro et al., 2007). In the present study, we show the results of purification and characterization of hyaluronidase enzyme from P. motoro venom. 2. Materials and methods 2.1. Venom and animals Specimens of P. motoro were collected from Crixa´ s-Ac-u´ River (Goia´ s, Brazil). The entire sting was removed with bistouries, lyophilized and scraped. The collected material was macerated and dissolved in phosphate buffer 50 mM, pH 7.0, containing 0.15 M NaCl and immediately centri- fuged at 5000g for 10 min. Venom was stored at À20 1C until use. 2.2. Estimation of protein concentration Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as a standard. 2.3. Assay of hyaluronidase enzyme activity Hyaluronidase enzyme activity was determined by the method described by Ferrante (1956), ARTICLE IN PRESS M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–1067 1061
  3. 3. modified by Poh et al. (1992). The assay mixture contained 200 ml acetate buffer 0.2 M, pH 6.0, containing 0.15 M NaCl, 50 ml hyaluronic acid (0.5 mg/ml in acetate buffer) and 50 ml enzyme in acetate buffer. The mixture was incubated for 15 min at 37 1C and the reaction was stopped by the addition of 500 ml of 2.5% (w/v) acetyltrimethy- lammonium bromide in 2% (w/v) NaOH. After 10 min, the absorbance of each reaction mixture was read at 400 nm. Specific activity was expressed as National Formulary Units (NFU), which is defined as the amount of enzyme required to hydrolyze 0.255 mg of the hyaluronic acid per minute. 2.4. SDS-polyacrylamide gel electrophoresis SDS-PAGE (12%) was carried out under dena- turing conditions according to the method described by Laemmli (1970). After electrophoresis, gel was silver stained as described by Blum et al. (1987). Molecular weight standards from 97.4 kDa (phos- phorylase B) and 66 kDa (BSA) were used. 2.5. Enzyme purification The crude P. motoro venom (0.38 mg) was loaded on a Sephacryl S-100 column (2.5 Â 48 cm) pre- viously equilibrated with 50 mM phosphate buffer, pH 6.0, containing 100 mM NaCl, and eluted with the same buffer at a flow rate of 40 ml/h. Fractions of 2.0 ml were collected and monitored at 280 nm. Fractions showing the highest hyaluronidase activ- ity were pooled, dialyzed and applied directly onto a SP-Sepharose column (1.5 Â 13 cm) equilibrated with 20 mM phosphate buffer, pH 6.0, and eluted at a flow rate of 60 ml/h. Fractions of 3.0 ml were collected and monitored at 280 nm. The column was washed with the same buffer and eluted with a linear gradient of 0–1.0 M NaCl. Fractions contain- ing hyaluronidase activity were pooled, dialyzed, lyophilized and stored at À20 1C. 2.6. Molecular weight determination The molecular weight of the purified hyaluroni- dase was estimated by gel filtration chromatography according to the method of Andrews (1962) on calibrated columns (2.5 Â 48 cm) of Sephacryl S- 100, using 50 mM phosphate buffer, pH 6.0 (con- taining 100 mM NaCl), at a flow rate of 40 ml/h. Void volume (Vo) of the column was determined by using blue dextran (1 mg/ml in equilibration buffer). Ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa) were used as standard proteins for obtaining the calibration curve. A calibration curve was obtained by plotting Ve/Vo (KAV) against their respective logarithmic molecular weights. 2.7. Enzyme characterization The effect of pH on enzyme activity was determined by varying the pH of the reaction mixtures using 100 mM phosphate–citrate buffer (pH 2.5–7.0). The effect of temperature on enzy- matic activity was determined at pH 4.2, in the range of 20–50 1C. The effect of temperature on enzyme stability was determined after preincubation at 20, 30 and 40 1C for 5–30 min. The effects of metallic ions and some compounds on hyaluroni- dase activity were determined after preincubation at 4 1C for 15 min. Km was determined from the Michaelis–Menten plot using Origin 7.0 program by measuring the initial rate of hyaluronic acid hydrolysis using a range of 2.5–25 mg/ml. 3. Results 3.1. Purification of hyaluronidase A two-step protocol was standardized for hyalur- onidase purification. The first step involved the Sephacryl S-100 gel filtration chromatography, which fractionated P. motoro venom gland extract into two peaks of proteins (Fig. 1A). Fractions with hyaluronidase activity were pooled and concen- trated by lyophilization. Only 3.62% of the activity loaded onto the column was recovered in the pooled fraction. The second step involved the SP-Sepharose ion-exchange chromatography and resolved into one peak of protein. The peak containing hyalur- onidase activity was eluted with a linear gradient of NaCl (Fig. 1B). In this step 80% of the enzyme loaded onto the column was recovered. A summary of the purification procedure is given in (Table 1). The enzyme was purified to 366.4-fold with a yield of 2.90%, having a specific activity of 1.33 Â 108 NFU/min mg of protein. SDS-PAGE showed that the enzyme migrated as a single band (Fig. 2). Molecular weight of the hyaluronidase was esti- mated by gel filtration on Sephacryl S-100 using standard protein molecular weight markers and it was found to be approximately 79 kDa (Fig. 3). ARTICLE IN PRESS M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–10671062
  4. 4. ARTICLE IN PRESS 20 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 50 100 150 200 250 Absorbanceat280nm() Fraction number HyaluronidaseactivityNFU/mL(-------) 0 0.00 0.01 0.02 0.03 0.04 0.05 0 50 100 150 200 250 0.0 0.2 0.4 0.6 0.8 1.0 Absorbanceat280nm() Fraction number HyaluronidaseactvityNFU/mL(------) NaCl(0-1.0M) 30 40 50 60 70 80 10 20 30 40 50 Fig. 1. Isolation of hyaluronidase from P. motoro venom. (A) Elution profile from Sephacryl S-100 chromatography. The column (2.5 Â 48 cm) was eluted with 50 mM phosphate buffer, pH 6.0, containing 100 mM NaCl at a flow rate of 40 ml/h, and 2 ml fractions were collected. Protein elution was monitored at 280 nm (———) and hyaluronidase activity at 400 nm (- - - - - -). Fractions having the hyaluronidase activity (dotted line) were pooled, concentrated and applied onto SP-Sepharose columns for further fractionation. (B) Elution profile from SP-Sepharose column chromatography. The column (1.5 Â 13 cm) was equilibrated with 20 mM phosphate buffer, pH 6.0, at a flow rate of 60 ml/h, and 2 ml fractions were collected. The column was washed with the same buffer and eluted with a linear gradiet of 0–1.0 M NaCl. Table 1 Summary of purification of hyaluronidase from P. motoro venom Purification step Total protein (mg) Total activity (NFU/ml) Specific activity (NFU/mg) Purification (fold) Yield (%) Crude venom 380 138 Â 103 3.63 Â 105 1 100 Sephacryl S-100 0.69 5 Â 103 7.24 Â 106 19.9 3.62 SP-Sepharose 0.03 4 Â 103 1.33 Â 108 366.4 2.90 M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–1067 1063
  5. 5. 3.2. Biochemical characterization The pH activity profile of purified hyaluronidase was determined in a pH range from 2.5 to 7.0 using phosphate/citrate buffer. The enzyme had a typical bell-shaped profile covering a broad pH range and an optimal pH of 4.2 (Fig. 4A). The influence of temperature on hyaluronidase activity was deter- mined between 4 and 50 1C at pH 4.2. The optimal temperature for hyaluronidase activity was 40 1C and the activity decreased significantly above 40 1C (Fig. 4B). The enzyme was stable for at least 30 min when incubated at 20 and 30 1C, but lost 70% of the activity at 40 1C (Fig. 4C). The effect of varying con- centrations of hyaluronic acid on the initial velocity of the hyaluronidase showed a typical hyperbolic saturation curve (Fig. 5). The Km (4.91 mg/ml) and Vmax (2.02 U/min) values were calculated from the Michaelis–Menten plot. The activity of the purified hyaluronidases was tested in presence of metal ions and some chemical compounds (Table 2). No considerable effect was observed with Ca2+ , Mg2+ , Zn2+ and Hg2+ , whereas Ni+ , Fe2+ and Cu2+ reduced activity by 25% approximately. b-Mercaptoethanol had a slight effect on enzyme activity, whereas heparin (0.05 IU) inhibited hyaluronidase activity by 20%. 4. Discussion In this study with P. motoro venom extract, we found that two-step fractionation on Sephacryl S- 100 column and SP-Sepharose column resulted in the purification of a protein with hyaluronidase activity. The final yield of 2.90% obtained and activity of 4 Â 103 NFU/ml will signify the difficult associated with working with this enzyme. Similar results were described by Xu et al. (1982) working with hyaluronidase from Agkistrodon acutus snake venom. However, Poh et al. (1992) recovered 57% hyaluronidase from Synanceja horrida stonefish venom and Pessini et al. (2001) recovered 43.6% hyaluronidase from Tityus serrulatus scorpion venom. Polyacrylamide gel electrophoresis showed that hyaluronidase purified from P. motoro migrated as a single band with an estimated molecular mass of 79 kDa, and exists as monomer. Most of the hyaluronidase described in the literature appears as a monomer and varies considerably between organisms. Molecular weight of hyaluronidase from Heterometrus fulvipus (Ramanaiah et al., 1990) was in a similar range, while proteins with 33, 52 and 116 kDa have been described from A. acutus (Xu et al., 1982), T. serrulatus (Pessini et al., 2001) and ARTICLE IN PRESS Fig. 2. SDS-PAGE of the purified P. motoro hyaluronidase. (Lane 1) Molecular weight markers. (Lane 2) Crude P. motoro venom. (Lane 3) Purified enzyme after Sephacryl S-100 chroma- tography. (Lane 4) Purified enzyme after SP-Sepharose chroma- tography. 0.05 1.0 1.2 1.4 1.6 1.8 2.0 MolecularWeightLog KAV Hyaluronidase Ovoalbumin Chymotrypsinogen Ribonuclease 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 Fig. 3. Determination of molecular mass of the purified hyaluronidase from the venom of P. motoro by gel filtration chromatography. The molecular weight of the purified hyalur- onidase was estimated by gel filtration chromatography on calibrated columns (2.5 Â 48 cm) of Sephacryl S-100, using 50 mM phosphate buffer, pH 6.0 (containing 100 mM NaCl), at a flow rate of 40 ml/h. Void volume (Vo) of the column was determined by using blue dextran (1 mg/ml in equilibration buffer), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa). A calibration curve was obtained by plotting Ve/Vo against their respective logarithmic molecular weights. M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–10671064
  6. 6. Streptococcus agalactiae (Ozegowski et al., 1994), respectively. The optimal pH for the enzyme activity (4.2) was similar to that found for hyaluronidase from a variety of organisms. The optimal pH for hyalur- onidase activity is usually in the range of 3.5 and 6.5 (Xu et al., 1982; Poh et al., 1992; Ozegowski et al., 1994; Morey et al., 2006; Nagaraju et al., 2007). The optimum temperature was found to be 37 1C at pH 4.2, and it is in agreement with hyaluronidase from Palamneus gravimanus (Morey et al., 2006) and Hippasa partita (Nagaraju et al., 2007). Thermo- stability is considered an important and useful criterion for enzyme characterization. The hyalur- onidase from P. motoro was stable for at least 30 min when incubated at 20 and 30 1C, but retained only 30% of the activity after incubation at 40 1C. ARTICLE IN PRESS 2.0 0 500 1000 1500 2000 2500 3000 3500 4000 Hyaluronidaseactivity(NFU/ml) pH 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 Hyaluronidaseactivity(NFU/ml) Temperature (°C) 0 20 30 40 50 60 70 80 90 100 110 (20°C) (30°C) (40°C) Relativeactivity(%) Time 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 10 20 30 40 50 5 10 15 20 25 30 Fig. 4. Characterization biochemistry of P. motoro hyaluronidase. (A) Profile of the pH optimum for hyaluronidase activity. (B) Profile of the temperature optimum for hyaluronidase activity. (C) Temperature–stability profile of hyaluronidase activity purified. 0 0.6 0.8 1.0 1.2 1.4 1.6 1.8 V(NFU/min) 5 10 15 20 25 Hyaluronic acid (µg/mL) Fig. 5. Michelis–Menten plot of the hyaluronidase activity with the substrate hyaluronic acid. Experiments were performed at 40 1C and pH 4.2. M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–1067 1065
  7. 7. The purified hyaluronidase from P. motoro showed Michaelis–Menten-type kinetics with hya- luronic acid as substrate. The Km of 4.91 mg/ml indicates that the enzyme has comparatively high affinity for hyaluronic acid compared with other hyaluronidases. This value was substantially lower than those reported for P. gravimanus (47.61 mg/ml) (Morey et al., 2006), T. serrulatus (69.7 mg/ml) (Pessini et al., 2001), S. agalactiae (81.9 mg/ml) (Ozegowski et al., 1994) and S. horrida stonefish (709 mg/ml) (Poh et al., 1992). As reported from studies on other hyaluronidase, a concentration as low as 10mM of some metal ions could affect enzyme activity. The poor inhibition by Hg2+ and b-mercaptoethanol, compounds that usual- ly react with cystein, led us to hypothesize about the absence of these amino acids in the catalytic site of the enzyme. Similar results are found by hyaluronidase from P. gravimanus (Morey et al., 2006). P. motoro purified enzyme is inhibited by heparin as described by hyaluronidase from A. acutus venom (Xu et al., 1982), H. fulvipus scorpion venom (Ramanaiah et al., 1990) and P. gravimanus (Morey et al., 2006). In conclusion, this study presents the first purification of a hyaluronidase from P. motoro sting. This enzyme shows similar characteristics as enzymes from venom of different organisms and exhibited high affinity for hyaluronic acid. Further structural and functional analyses might provide an insight for the better understanding of the role of this enzyme in envenomation by P. motoro. Acknowledgments This work was supported by a biotechnology research grant to C.J.U. (CNPq, CAPES and FUNAPE/UFG). M.R.M. was supported by Uni- versidade Cato´ lica de Goia´ s (CEPB). The authors thank Dr. Joa˜ o Luiz da Costa Cardoso and Dr. Vidal Haddad Jr. (Hospital Vital Brazil, Instituto Butantan), and Dra Ka´ tia Cristina Barbaro (La- borato´ rio de Imunopatologia, Instituto Burantan) for valuable suggestions during this study. References Andrews, P., 1962. Estimation of molecular weights of proteins by gel filtration. Nature 196, 36–39. Barbaro, K.C., Lira, M.S., Malta, M.B., Soares, S.L., Garrone Neto, D., Cardoso, J.L.C., Santoro, M.L., Haddad Jr., V., 2007. Comparative study on extracts from the tissue convering the stingers of freswater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays. Toxicon, in press. Blum, H., Beier, H., Gross, H., 1987. Improvised silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99. Caras, R., 1974. The venomous fish. In: Caras, R. (Ed.), Venomous Animals of the World. New York, pp. 103–116. Carvalho, M.R., Lovejoy, N.R., Rosa, R.S., 2003. Family Potamotrygonidae (river stingrays). In: Reis, R.E., Kullander, S.O., Ferraris, C.J. (Eds.), Check List of the Freshwater Fishes of South and Central America. Edipucrs, Porto Alegre, pp. 22–28. Castex, M.N., 1965. Clı´nica y terapeutica de la enfermedad paratrygonica. Rev. Asoc. Med. Argent. 79, 547–557. Castex, M.N., Loza, F., 1964. Etiologia de la enfermedad paratrygo´ nica, estudio anato´ mico, histolo´ gico y funcional del aparato agressor de la raya fluvial americana (gen. Potamotrygon). Rev. Asoc. Med. Argent. 78, 314–324. Charvet-Almeida, P., Arau´ jo, M.L.G., Rosa, R.S., Rinco´ n, G., 2002. Neotropical freshwater stingrays: diversity and con- servation status. Shark News 14, 47–51. Clark, R.F., Girard, R.H., Rao, D., Ly, B.T., Davis, D.P., 2007. Stingray envenomation: a retrospective review of clinical presentation and treatment in 119 cases. J. Emerg. Med. 33, 33–37. ARTICLE IN PRESS Table 2 Inhibitor effects in the activity of P. motoro hyaluronidase Compound (10 mM) Enzymatic activity (NFU/ml) Relative activity (%) Inhibition (%) Control 4305.9176.9 100 0 CaCl2 3926.257114.14 91.2 8.8 FeSO4 3220.97122.12 74.8 25.2 HgCl2 3712.87111.9 86.2 13.8 MgCl2 4046.09773.00 93.9 6.4 CuSO4 3044.51719.6 70.7 29.3 MnSO4 2149.9719.62 49.9 50.1 ZnSO4 3656.6712.84 84.9 15.1 b-Mercaptoetanol 4278.85719.64 99.3 0.7 Heparin (0.05 UI) 3425.43778.00 79.5 20.5 M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–10671066
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