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Effect of crotapotin on the biological activity of Asp49 and Lys49 phospholipases A2 from Bothrops snake venoms
Comparative Biochemistry and Physiology, Part C 138 (2004) 429 – 436 www.elsevier.com/locate/cbpc
Effect of crotapotin on the biological activity of Asp49 and Lys49 phospholipases A2 from Bothrops snake venoms A.L. Cecchinia,b, A.M. Soaresc, R. Cecchinid, A.H.C. de Oliveirae, R.J. Wardf, J.R. Gigliob, E.C. Arantesa,* a
Depto de Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, USP, Brazil b Depto de Bioquı´mica e Imunologia, Faculdade de Medicina de Ribeira˜o Preto, USP, Brazil c Unidade de Biotecnologia, UNAERP-Ribeira˜o Preto, Brazil d Depto de Cieˆncias Patolo´gicas-CCB, UEL, Brazil e Depto de Biologia Celular e Molecular e Bioagentes Patogeˆnicos, Faculdade de Medicina de Ribeira˜o Preto, USP, Brazil f Depto de Quı´mica, Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, USP, Ribeira˜o Preto-SP, Brazil Received 2 April 2004; received in revised form 7 July 2004; accepted 14 July 2004
Abstract Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A2 (PLA2s). Asp49-PLA2s are catalytically active, whereas Lys49-PLA2s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA2 (CB), is a PLA2-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA2s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA2s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction. D 2004 Elsevier Inc. All rights reserved. Keywords: Crotapotin; Bothrops; Crotoxin; Phospholipase A2; Snake venom; Circular dichroism; Infrared spectrum; Edema; Myotoxicity; Liposome disruption
1. Introduction
Abbreviations: BthTX-I, Lys49 bothropstoxin-I from B. jararacussu; BthTX-II, Asp49 bothropstoxin-II from B. jararacussu; MjTX-II, Lys49 myotoxin-II from B. moojeni; PrTX-I, Lys49 piratoxin-I from B. pirajai; PrTX-III, Asp49 piratoxin-III from B. pirajai; CA, crotapotin; PLA2, phospholipase A2; CB, crotoxin PLA2; CK, creatine kinase; CD, circular dichroism; FT-IR, Fourier Transformed Infrared Spectroscopy. * Corresponding author. Tel.: +55 16 6024275; fax: +55 16 6332960. E-mail address: [email protected] (E.C. Arantes). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.07.010
The local effects caused by Bothrops venoms include edema, pain, hemorrhage and necrosis, which may result in prolonged or permanent disability (Moura da Silva et al., 1991; Ferreira et al., 1992). Furthermore, the ischaemia evoked by edema (Chapman, 1968) may aggravate the venom-induced lesion. Myonecrosis is the most striking local effect caused by Bothrops snake venoms, and muscular atrophy is a common sequella following bites by this genus (Queiro´z et al., 1984; Milani et al., 1997; Jorge et al., 1999). Since myonecrosis is caused by a direct action of
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the venom on muscle fibers (Queiro´z et al., 1984), the pathogenesis and prevention of muscle lesion have been extensively studied (Melo and Suarez-Kurtz, 1988; Melo et al., 1993; Soares et al., 1997; Melo and Ownby, 1999; Fernandez et al., 2000; Oshima-Franco et al., 2000). Most of the myotoxic activity of snake venoms is due to phospholipase A2 (PLA2) toxins, among which Asp49 variants are catalytically active (Ownby et al., 1997) whereas others, although having a PLA2 conformation, have little or no enzymatic activity due to a substitution of lysine for aspartic acid at position 49 (Maraganore et al., 1984; Homsi-Brabdeburgo et al., 1988; Cintra et al., 1993; Johnson and Ownby, 1993; Selistre de Arau´jo et al., 1996a; Pereira et al., 1998; Soares et al., 1998, 2000a; Rodrigues et al., 2000). Phospholipase A2 toxins are also implicated directly and indirectly in the formation of edema (Lomonte et al., 1993). The exact mechanism of edema formation is, however, still under investigation. Administration of a chemical agent that is antagonistic to phospholipases A2 reduces the overall effect of the venom action, and therefore decreases the need for extensive therapy. Crotoxin is a heterodimeric protein composed of a basic Asp49-PLA2 (pI ~8.2) and an acidic protein named crotapotin (pI ~3.4), CA and CB standing for crotoxin acid and crotoxin base, respectively (Breithaupt et al., 1974). This PLA2 is catalytically active and responsible for the myotoxic activity of crotoxin observed in vivo (Gopalakrishnakone et al., 1984). Crotapotin consists of three polypeptide chains linked together by disulphide bridges and is thought to act as a chaperone protein for PLA2 (Bon et al., 1979). Although CA is enzymatically and pharmacologically inactive (Haberman and Breithaupt, 1978; Bon et al., 1979; Verheij et al., 1980; Gopalakrishnakone et al., 1984), it enhances the toxicity of PLA2 (Bon, 1982) and also seems to play a role in oligomer assembly, protein transport, DNA replication and mRNA turnover (Ellis, 1990). CA inhibits the formation of carrageenin-induced rat paw edema, probably by interacting with secreted PLA2s generated during the inflammatory process (Landucci et al., 1995). CA also inhibits edema induced by different snake venoms (Landucci et al., 2000). We report here a study on the inhibitory effect of crotapotin on mouse paw edema, myonecrosis and liposome membrane disruption induced by isolated PLA2s (Asp49 and Lys49) from three different kinds of Bothrops snake venoms (BthTX-I and BthTX-II from Bothrops jararacussu, PrTX-I and PrTX-III from Bothrops pirajai and MjTX-II from Bothrops moojeni), focusing on probable modifications in the secondary structure of these myotoxins or simple activity blockage due to ionic and/or hydrophobic interactions with strategic amino acid residues. In order to correlate the biological effects with structural modifications, we have also assayed the enzymatic activity, infrared spectroscopy and circular dichroism of the assayed PLA2s in the presence and absence of CA.
2. Materials and methods 2.1. Phospholipase A2 activity CA and PLA2s were isolated and purified as previously described (Soares et al., 2001). PLA2 activity was evaluated potentiometrically, using egg yolk as substrate as described by de Hass et al. (1968). 2.2. Myotoxic activity The assay for determination of plasma creatine kinase (CK) activity was carried out using the CK-UV kinetic kit from Sigma. Toxins (50 Ag/dose) and CA (100 Ag/dose), molar ratio CA/toxin ~3, were injected intramuscularly in the gastrocnemius muscle of 18–22-g male Swiss mice (50 Al, n=6). Animals used as negative controls were injected with sterile saline. After 3 h, a blood sample was collected from the tail in heparinized capillary tubes and centrifuged for plasma separation (Soares et al., 2000b,c). CK activity was determined using 4 Al of plasma according to the manufacturer’s instructions. The enzyme activity was expressed in U/l, 1 U being defined as the amount of enzyme that produces 1 Amol of NADH/min under the conditions of the assay. 2.3. Edema-inducing activity Groups of five male Swiss mice (18–22 g) were injected subcutaneously in the subplantar region with 50 Al of toxins (5 Ag/paw) or toxins plus CA (10 Ag/paw), molar ratio CA/ toxin ~3. At 30 min and 1 h time intervals, the thickness of the paw was measured with a low pressure spring caliper (Mitutoyo, Japan) as an index of edema (Soares et al., 2000a,b). The thickness of the paw without injection was subtracted from all the values and its increase was expressed as a percentage. 2.4. Circular dichroism Far UV circular dichroism spectra (190–250 nm) were measured with a JASCO 810 (JASCO, Tokyo, Japan) using 1 mm path length cuvettes and a protein concentration of 250 Ag/ml for all myotoxins. For mixtures of CA and PLA2, the concentration of CA was 375 Ag/ml and the total protein concentration was 525 Ag/ml. In all measurements, five spectra were collected, averaged, and corrected by subtraction of a buffer blank. 2.5. Release of entrapped fluorescent markers from liposomes Membrane damaging activity was evaluated by the release of the liposome entrapped self-quenching fluorescent dye calcein. The loss of liposome membrane integrity results in release of the fluorophore with a consequent
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part C 138 (2004) 429–436 Table 1 Myotoxicity and enzymatic activities of myotoxic phospholipases A2 from Bothrops venoms in the absence and presence of crotapotin (CA) (1:2 w/w) Samples
Myotoxicity CK (U/l)
Phospholipasic A2 activity (U/mg)
Saline CA BthTX-I BthTX-I+CA BthTX-II BthTX-II+CA PrTX-I PrTX-I+CA PrTX-III PrTX-III+CA MjTX-II MjTX-II+CA
290.1 355.3 2200.4F133a 1303.7F128a b 3200.1F257a 1753.9F205a b 2253.6F132a 1030.3F164a b 2915.6F205a 1598.1F213a b 1913.4F161a 930.2F146a b
– 0.00 0.00 0.00 51.33 48.97 0.00 0.00 47.83 46.59 0.00 0.00
a b
Pb0.01 compared to the saline values. Pb0.01 compared to its PLA2 pair.
increase in the fluorescence signal (de Oliveira et al., 2001). Liposomes composed of a 9:1 molar ratio of egg yolk phosphatidylcholine (EYPC):dimyristoyl phosphatidic acid (DMPA) were prepared by reverse phase evaporation in a buffer (150 mM NaCl, 25 mM HEPES pH 7.0) containing 25 mM calcein (Sigma). The liposomes were passed through a 400-nm polycarbonate filter (Nucleopore, Pleasanton, CA, USA), and applied to a Sepharose 6 B column (18 cm) to separate the liposome entrapped calcein from the free calcein. Proteins and liposomes were mixed to a final toxin/lipid molar ratio of 1:200, and the kinetics of membrane damage was monitored by the increase in fluorescence emission at 520 nm with excitation at 490 nm following addition of the toxins. The fluorescence signal was expressed as the percentage of total calcein released following addition of 5 mM Triton X-100. The concentrations of toxins and CA were 3 and 4.5 Ag/ml, respectively, and Ca2+ was added to a final concentration of 2 mM and incubated for 30 min prior to assay.
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3. Results Table 1 presents the results obtained from the CK levels in mouse plasma and from the enzymatic activity assays measured using egg yolk as substrate. It is evident that there is no correlation between myotoxicity and phospholipase activity and that the presence of CA in the medium does not affect the hydrolytic activity of Asp49-PLA2s. The dose– effect curves for myotoxic and edema-forming activities are shown in Fig. 1. The myotoxic activity of PLA2s from the venom of Bothrops snakes promotes an increase of CK levels in mouse plasma. In the presence of CA, there was a significant decrease in the CK levels induced by PLA2s that indicates a 40% to 54% inhibition of the myotoxic effect. Bothrops phospholipases A2 also cause edema when injected into mouse paw (Fig. 2). CA significantly inhibited the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%) and PrTX-III (35%). In the case of
2.6. Fourier Transformed Infrared Spectroscopy analysis FT-IR Spectra were obtained on a Prote´ge´—460 MagnaIR Technology. BthTX-II and CA were previously incubated for 30 min and lyophilized before the assay. Approximately 100 Ag of the sample were mixed with 5 mg KBr (FT-IR grade) and analyzed along a 500–4000-cm1 range at a 4cm1 resolution. The computer program used to collect the data was Omnic and the results imported from Omnic to Origin 6. 2.7. Statistical analysis Results are presented as mean F standard error of mean (S.E.M.) of values obtained with the indicated number of animals. The statistical significance of differences between groups was evaluated using Student’s unpaired t-test. A P value b0.05 was considered to indicate significance.
Fig. 1. Representative dose-dependence inhibitory effect of crotapotin (CA) on myotoxic (A) and edema-inducing (B) activities of BthTX-II (*Pb0.01).
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Fig. 2. Effect of crotapotin (CA) on mouse paw edema induced by PLA2s isolated from Bothrops jararacussu BthTX-I (A) and BthTX-II (B), B. pirajai PrTX-I (C) and PrTX-III (D) and B. moojeni MjTX-II (E). The edema inducing response to crotapotin ( ) and PLA2s in the absence (E) or presence (z) of crotapotin is shown. The results represent the meanFS.E.M. of 12 animals. *Pb0.05, **Pb0.01 compared with the respective PLA2 (E).
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MjTX-II, the 10% inhibitory effect reduced the edema to the levels observed in the controls used in this assay (saline and CA alone). The edema inducing experiment was performed over a 1 h period, the highest response appearing at 30 min. Sixty minutes after injection, the swollen paw was returning to normal. Possible secondary structural changes in the toxins on incubation with CA were evaluated with circular dichroism (CD) spectroscopy. The results for the mixture of PLA2 and
CA shown in Fig. 3 revealed that the sum of the two individual CD spectra of PrTX-I and CA were similar to the spectra of an equimolar mixture of the two proteins. This result indicates that the association between PrTX-I and CA does not change the secondary structure of either the toxin or the CA. Similar results were observed in the experiments using BthTX-I, BthTX-II and MjTX-II (data not shown). Amongst the Lys49-PLA2s tested, MjTX-II presented a significantly increased membrane damaging activity in
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Fig. 3. Far UV circular dichroism spectra of PrTX-I, CA and an equimolar mixture of both proteins. Spectra of PrTX-I (n) and CA ( ) alone, the mixture at a 1:1 molar ratio (4), and the calculated sum of the individual spectra for the PrTX-I and CA (E) are shown.
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comparison with the other toxins, as evaluated by the release of entrapped calcein from liposomes (Fig. 4). In all cases, the membrane damaging activity of the toxins was reduced in the presence of CA, and although this reduction was slight, the effect was highly reproducible and statistically significant ( Pb0.05 in a paired t-test). FT-IR analysis showed that each protein has its own absorbance profile and that after complexation of CA with BthTX-II, the analysis reveals a third spectrum, suggesting an interaction among the structural groups at specific wavenumbers. In Fig. 5, the arrows show the presence of chemical groups in BthTX-II (Fig. 5A) and CA (Fig. 5B) that could interact with each other, whose signals decreased after complexation (Fig. 5C). Complexation of CA with other toxins showed similar spectra (data not shown). A band originating from COOH stretching vibrations, situated at 2856.77 cm1 for CA and 2931.33 for BthTX-II, refers to stretching vibrations of hydrocarbon aliphatic groups, e.g. CH2 and CH3 (Fig. 5A,B).
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necrosis and polymorphonuclear infiltration in the presence of CA 3 h after injection. This inhibitory action was most clearly observed when CA was incubated with MjTX-II, and in this case the morphological alterations were similar to the control (data not shown). Inhibition of edema by the association of CA with other PLA2s had been reported (Landucci et al., 2000). In our study, we observed a greater inhibition of the edema-forming activity when crotapotin was incubated with Asp49-PLA2s (30% inhibition) than with Lys49-PLA2s (13% inhibition), although we could not observe any significant inhibition of Asp49-PLA2 catalytic activity in the presence of CA. CA, however, inhibits the phospholipase activity of its natural complex, CB, and increases its lethality. It is known that crotoxin is formed by complexation of one molecule of CB with one molecule of CA, although free PLA2 can still be identified in the crude venom. Numerous natural inhibitors of toxic PLA2s have recently been isolated from the blood of a venomous snake and a marsupial (Soares et al., 1997, 2003) and from some plant extracts (Borges et al., 2000). Phospholipase inhibitors play an important role in the neutralization of neurotoxins and myotoxins, and some of these inhibitors may be useful in the future for envenomation treatment. Edema formation is a common feature of the inflammatory response and is dependent on a synergism between mediators that increase vascular permeability and those that increase blood flow (Williams and Morley, 1973; Williams and Peck, 1977). Histological analysis of the paw 30 min and 1 h after injection was performed (data not shown) and the pattern of the lesion showed a clear inflammation process with the absence of polymorphonuclear red blood cells and epidermal damage or necrosis. Since the paw returns to
4. Discussion Edema and myonecrosis are common effects of envenomation by snake bites, especially those of the genus Bothrops. The present study shows that a protein from the venom of a Crotalidae snake (C. d. terrificus), crotapotin (CA), is able to inhibit edema-inducing, myotoxic effects and liposome membrane disruption evoked by phospholipases A2 from the venom of several Bothrops snakes. In general, the combination of CA with PLA2 prior to injection resulted in significant reductions in both edema and myonecrosis. CA inhibited around 40–50% of the myotoxicity caused by PLA2s, as measured by plasma CK levels (Table 1). Histological analysis (data not shown) confirms the CK results, showing a decrease in muscle fiber
Fig. 4. Membrane damaging activities of the toxins (3 Ag/ml) alone and after incubation with CA (4.5 Ag/ml). The loss of liposome membrane integrity results in release of the fluorophore. The fluorescence signal was expressed as the percentage of total calcein released following addition of 5 mM Triton X-1000. *Pb0.05, compared with the respective PLA2.
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Fig. 5. Fourier Transform Infrared Spectroscopy (FT-IR) was obtained for BthTX-II (A), crotapotin (B) and the complex BthTX-II+CA (C). The arrows show the modifications observed on both spectra after formation of the complex. Note that these modifications are evidenced by alterations in the wavenumber parameters.
normal after 3 h, we suggest that the inflammation process is restricted to the area of injection. The presence of aspartic acid at position 49 is crucial for calcium binding to the Asp49-PLA2s and Ca2+ is essential for catalytic activity (Scott et al., 1992). Despite the lack of an aspartic acid residue at position 49, the Lys49-PLA2 proteins are potent inducers of myonecrosis by one or more unknown mechanisms (Homsi-Brabdeburgo et al., 1988; Johnson and Ownby, 1993). Selistre de Arau´jo et al. (1996a) observed that Lys49-PLA2s comprise a highly conserved protein family very distinct from the Asp49-PLA2 protein family, and comparison of Lys49-PLA2 amino acid sequences reveals a high level (70–95%) of identity. In contrast, amino acid identity within the Asp49 group can be as low as 47% in the case of the Asp49-PLA2 from Crotalus adamanteus (Selistre de Arau´jo et al., 1996b). Despite these differences in the degree of amino acid conservation within the Asp49-PLA2s and the Lys49-PLA2s, the results reported here show that there are interactions between crotapotin and both Asp49- and Lys49-PLA2s, from the venoms of different species.
Although no indication of structural modifications was detectable by either CD or FT-IR analysis, an alteration in absorbance at wavenumber=2856 cm1 in the FT-IR spectrum for the carboxyl terminal groups of crotapotin suggests an ionic interaction, and similar alterations for the aliphatic carbon skeleton indicate hydrophobic interactions at wavenumber 2931 cm1 for BthTX-II and 2924 cm1 for crotapotin (Fig. 5). The differences observed in Fig. 5C are in the range 2873.40–3064.63 cm1, where those groups should vibrate. There is a clear decrease of the stretching vibration, suggesting a steric impeachment for those groups to move freely. This may be explained by interactions with other PLA2s, forming homodimers (da Silva Giotto et al., 1998). It has been reported that CA is a PLA2-like protein (Bouchier et al., 1991), so it is likely to interact with other PLA2s as it interacts with the CB in the C. d. terrificus venom to form the neurotoxin crotoxin. Analysis of infrared spectra allows us to state that there are interactions between CA and BthTX-II that involve carboxyl and aliphatic groups. The nature of the biochemical interactions between PLA2s and crotapotin remains to be established and may
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involve chemical impairment of some amino acid residues to properly play their functional role (Soares et al., 2001). In the liposome membrane disruption assay, the MjTX-II was shown to have increased activity when compared with other PLA2s tested. This may be a consequence of a peculiar amino acid sequence in the C-terminal loop region of this protein. Recent site-directed mutagenesis results with BthTX-I have demonstrated that alterations in the physical–chemical properties of this region have significant effects on the membrane damaging effect (Chioato et al., 2002). In this report, we have presented evidence that crotapotin inhibits the edema and myotoxic activities of several Bothrops PLA2, and this effect could be related to the prevention of membrane disorganization since liberation of calcein in the liposome assay was shown to be significantly decreased when PLA2 was incubated with CA. As our data point to blockage of strategic binding sites of BthTX-II by CA, rather than to detectable modifications of its secondary structure, it is plausible to assume that inhibition of liposome disruption is a consequence of a partial hindrance of binding to its membrane surface due to the above mentioned blockage, which would be equivalent to sitedirected mutagenesis of specific amino acids involved with the binding event (Chioato et al., 2002). Inhibition of PLA2 toxicity without inducing structural alterations would be required to definitively specify the myotoxic action of Lys49-PLA2s and Asp49-PLA2s. Previous experiments suggest that both hydrolytic activity and biological effects do not occur in the same catalytic site (Soares et al., 2001). Crotapotin displays a significant inhibitory effect on Bothrops PLA2s, oppositely to what happens with its natural Crotalus PLA2 counterpart, which is strongly potentiated by this acidic protein. Although CA potentiates CB at a 1:1 ratio (mol/ mol), excess CA inhibits its edema inducing, neurotoxic and anti-bacterial activities (Cecchini et al., 2004). Not surprisingly, C. d. terrificus venom displays excess PLA2s over CA, although these PLA2s are apparently CB homologous (work in preparation). More research is still necessary to fulfill the requirements for use of crotapotin for therapeutics of envenomation by Bothrops venoms or other PLA2-containing venoms. In addition, this work points to the use of CA as a promising tool to explore PLA2s structure and its relationship to biological activity. Acknowledgements The authors gratefully acknowledge the financial support from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), as well as the skillful technical assistance of Odete A.B. Cunha and Carlos A. Vieira.
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of neuwiedase, a non-hemorrhagic fibrin(ogen)olytic metalloprotease from Bothrops neuwidi snake venom. Arch. Biochem. Biophys. 381, 213 – 224. Scott, D.L., Achari, A., Vidal, J.C., Sigler, P.B., 1992. Crystallographic and biochemical studies of the (inactive) Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. J. Biol. Chem. 267, 22645 – 22657. Selistre de Arau´jo, H.S., White, S.P., Ownby, C.L., 1996a. cDNA cloning and sequence analysis of a lysine-49 phospholipase A2 myotoxin from Agkistrodon contortrix laticinctus snake venom. Arch. Biochem. Biophys. 326, 21 – 30. Selistre de Arau´jo, H.S., White, S.P., Ownby, C.L., 1996b. Sequence analysis of Lys49 phospholipase A2 myotoxins: a highly conserved class of proteins. Toxicon 34, 1237 – 1242. Soares, A.M., Rodriguez, V.M., Borges, M.H., Andria˜o-Escarso, S.H., Cunha, O.A.B., Homsi-Brandeburgo, M.I., Giglio, J.R., 1997. Inhibition of proteases, myotoxins and phospholipase A2 from Bothrops venoms by the heterometric protein complex of Didelphis albiventris opossum serum. Biochem. Mol. Int. 43, 1091 – 1099. Soares, A.M., Rodrigues, V.M., Homsi-Brandeburgo, M.I., Toyama, M.H., Lombardi, F.R., Arni, R.K., Giglio, J.R., 1998. Rapid procedure for isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: biochemical characterization myotoxic and edematogenic activity. Toxicon 36, 503 – 514. Soares, A.M., Guerra-Sa´, R., Rodrigues, V.M., Rodrigues-Simioni, L., Fontes, M.R.M., Giglio, J.R., 2000a. Structural and functional characterization of BnSP-7, a Lysine-49 myotoxin phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom. Arch. Biochem. Biophys. 378, 201 – 209. ˆ ngulo, Y., Lomonte, B., Gutie´rrez, Soares, A.M., Andria˜o-Escarso, S.H., A J.M., Marangoni, S., Toyama, M.H., Arni, R.K., Giglio, J.R., 2000b. Structural and functional characterization of a myotoxin I, a Lys49 phospholipase A2 homologue from Bothrops moojeni (caissaca) snake venom. Arch. Biochem. Biophys. 373, 7 – 15. Soares, A.M., Guerra-Sa´, R., Borja-Oliveira, C., Rodrigues, V.M., Rodrigues-Simioni, L., Rodrigues, V., Fontes, M.R., Lomonte, B., Gutie´rrez, J.M., Giglio, J.R., 2000c. Structural characterization of BnSP-7, a Lys49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom. Arch. Biochem. Biophys. 378, 201 – 209. Soares, A.M., Mancin, A.C., Cecchini, A.L., Arantes, E.C., Franc¸a, S.C., Gutie´rrez, J.M., Giglio, J.R., 2001. Effects of chemical modifications of crotoxin B, the phospholipase A2 subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities. Int. J. Biochem. Cell Biol. 33, 877 – 888. Soares, A.M., Marcussi, S., Franc¸a, S.C., Giglio, J.R., Ward, R.J., Arantes, E.C., 2003. Structural and functional analysis of BmjMIP, a phospholipase A2 myotoxin inhibitor protein from Bothrops moojeni snake plasma. Biochem. Biophys. Res. Commun. 302, 193 – 200. Verheij, H.M., Boffa, M.C., Rothen, C., Bryckaert, M., Verger, R., de Hass, G.H., 1980. Correlation of enzymatic activity and anticoagulant properties of phospholipase A2. Eur. J. Biochem. 112, 25 – 32. Williams, T.J., Morley, J., 1973. Prostaglandins as potentiators of increased vascular permeability in inflammation. Nature 246, 215 – 217. Williams, T.J., Peck, M.J., 1977. Role of prostaglandin-mediated vasodilatation in inflammation. Nature 270, 530 – 532.
Effect of crotapotin on the biological activity of Asp49 and Lys49 phospholipases A2 from Bothrops snake venoms A.L. Cecchinia,b, A.M. Soaresc, R. Cecchinid, A.H.C. de Oliveirae, R.J. Wardf, J.R. Gigliob, E.C. Arantesa,* a
Depto de Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, USP, Brazil b Depto de Bioquı´mica e Imunologia, Faculdade de Medicina de Ribeira˜o Preto, USP, Brazil c Unidade de Biotecnologia, UNAERP-Ribeira˜o Preto, Brazil d Depto de Cieˆncias Patolo´gicas-CCB, UEL, Brazil e Depto de Biologia Celular e Molecular e Bioagentes Patogeˆnicos, Faculdade de Medicina de Ribeira˜o Preto, USP, Brazil f Depto de Quı´mica, Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, USP, Ribeira˜o Preto-SP, Brazil Received 2 April 2004; received in revised form 7 July 2004; accepted 14 July 2004
Abstract Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A2 (PLA2s). Asp49-PLA2s are catalytically active, whereas Lys49-PLA2s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA2 (CB), is a PLA2-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA2s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA2s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction. D 2004 Elsevier Inc. All rights reserved. Keywords: Crotapotin; Bothrops; Crotoxin; Phospholipase A2; Snake venom; Circular dichroism; Infrared spectrum; Edema; Myotoxicity; Liposome disruption
1. Introduction
Abbreviations: BthTX-I, Lys49 bothropstoxin-I from B. jararacussu; BthTX-II, Asp49 bothropstoxin-II from B. jararacussu; MjTX-II, Lys49 myotoxin-II from B. moojeni; PrTX-I, Lys49 piratoxin-I from B. pirajai; PrTX-III, Asp49 piratoxin-III from B. pirajai; CA, crotapotin; PLA2, phospholipase A2; CB, crotoxin PLA2; CK, creatine kinase; CD, circular dichroism; FT-IR, Fourier Transformed Infrared Spectroscopy. * Corresponding author. Tel.: +55 16 6024275; fax: +55 16 6332960. E-mail address: [email protected] (E.C. Arantes). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.07.010
The local effects caused by Bothrops venoms include edema, pain, hemorrhage and necrosis, which may result in prolonged or permanent disability (Moura da Silva et al., 1991; Ferreira et al., 1992). Furthermore, the ischaemia evoked by edema (Chapman, 1968) may aggravate the venom-induced lesion. Myonecrosis is the most striking local effect caused by Bothrops snake venoms, and muscular atrophy is a common sequella following bites by this genus (Queiro´z et al., 1984; Milani et al., 1997; Jorge et al., 1999). Since myonecrosis is caused by a direct action of
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the venom on muscle fibers (Queiro´z et al., 1984), the pathogenesis and prevention of muscle lesion have been extensively studied (Melo and Suarez-Kurtz, 1988; Melo et al., 1993; Soares et al., 1997; Melo and Ownby, 1999; Fernandez et al., 2000; Oshima-Franco et al., 2000). Most of the myotoxic activity of snake venoms is due to phospholipase A2 (PLA2) toxins, among which Asp49 variants are catalytically active (Ownby et al., 1997) whereas others, although having a PLA2 conformation, have little or no enzymatic activity due to a substitution of lysine for aspartic acid at position 49 (Maraganore et al., 1984; Homsi-Brabdeburgo et al., 1988; Cintra et al., 1993; Johnson and Ownby, 1993; Selistre de Arau´jo et al., 1996a; Pereira et al., 1998; Soares et al., 1998, 2000a; Rodrigues et al., 2000). Phospholipase A2 toxins are also implicated directly and indirectly in the formation of edema (Lomonte et al., 1993). The exact mechanism of edema formation is, however, still under investigation. Administration of a chemical agent that is antagonistic to phospholipases A2 reduces the overall effect of the venom action, and therefore decreases the need for extensive therapy. Crotoxin is a heterodimeric protein composed of a basic Asp49-PLA2 (pI ~8.2) and an acidic protein named crotapotin (pI ~3.4), CA and CB standing for crotoxin acid and crotoxin base, respectively (Breithaupt et al., 1974). This PLA2 is catalytically active and responsible for the myotoxic activity of crotoxin observed in vivo (Gopalakrishnakone et al., 1984). Crotapotin consists of three polypeptide chains linked together by disulphide bridges and is thought to act as a chaperone protein for PLA2 (Bon et al., 1979). Although CA is enzymatically and pharmacologically inactive (Haberman and Breithaupt, 1978; Bon et al., 1979; Verheij et al., 1980; Gopalakrishnakone et al., 1984), it enhances the toxicity of PLA2 (Bon, 1982) and also seems to play a role in oligomer assembly, protein transport, DNA replication and mRNA turnover (Ellis, 1990). CA inhibits the formation of carrageenin-induced rat paw edema, probably by interacting with secreted PLA2s generated during the inflammatory process (Landucci et al., 1995). CA also inhibits edema induced by different snake venoms (Landucci et al., 2000). We report here a study on the inhibitory effect of crotapotin on mouse paw edema, myonecrosis and liposome membrane disruption induced by isolated PLA2s (Asp49 and Lys49) from three different kinds of Bothrops snake venoms (BthTX-I and BthTX-II from Bothrops jararacussu, PrTX-I and PrTX-III from Bothrops pirajai and MjTX-II from Bothrops moojeni), focusing on probable modifications in the secondary structure of these myotoxins or simple activity blockage due to ionic and/or hydrophobic interactions with strategic amino acid residues. In order to correlate the biological effects with structural modifications, we have also assayed the enzymatic activity, infrared spectroscopy and circular dichroism of the assayed PLA2s in the presence and absence of CA.
2. Materials and methods 2.1. Phospholipase A2 activity CA and PLA2s were isolated and purified as previously described (Soares et al., 2001). PLA2 activity was evaluated potentiometrically, using egg yolk as substrate as described by de Hass et al. (1968). 2.2. Myotoxic activity The assay for determination of plasma creatine kinase (CK) activity was carried out using the CK-UV kinetic kit from Sigma. Toxins (50 Ag/dose) and CA (100 Ag/dose), molar ratio CA/toxin ~3, were injected intramuscularly in the gastrocnemius muscle of 18–22-g male Swiss mice (50 Al, n=6). Animals used as negative controls were injected with sterile saline. After 3 h, a blood sample was collected from the tail in heparinized capillary tubes and centrifuged for plasma separation (Soares et al., 2000b,c). CK activity was determined using 4 Al of plasma according to the manufacturer’s instructions. The enzyme activity was expressed in U/l, 1 U being defined as the amount of enzyme that produces 1 Amol of NADH/min under the conditions of the assay. 2.3. Edema-inducing activity Groups of five male Swiss mice (18–22 g) were injected subcutaneously in the subplantar region with 50 Al of toxins (5 Ag/paw) or toxins plus CA (10 Ag/paw), molar ratio CA/ toxin ~3. At 30 min and 1 h time intervals, the thickness of the paw was measured with a low pressure spring caliper (Mitutoyo, Japan) as an index of edema (Soares et al., 2000a,b). The thickness of the paw without injection was subtracted from all the values and its increase was expressed as a percentage. 2.4. Circular dichroism Far UV circular dichroism spectra (190–250 nm) were measured with a JASCO 810 (JASCO, Tokyo, Japan) using 1 mm path length cuvettes and a protein concentration of 250 Ag/ml for all myotoxins. For mixtures of CA and PLA2, the concentration of CA was 375 Ag/ml and the total protein concentration was 525 Ag/ml. In all measurements, five spectra were collected, averaged, and corrected by subtraction of a buffer blank. 2.5. Release of entrapped fluorescent markers from liposomes Membrane damaging activity was evaluated by the release of the liposome entrapped self-quenching fluorescent dye calcein. The loss of liposome membrane integrity results in release of the fluorophore with a consequent
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part C 138 (2004) 429–436 Table 1 Myotoxicity and enzymatic activities of myotoxic phospholipases A2 from Bothrops venoms in the absence and presence of crotapotin (CA) (1:2 w/w) Samples
Myotoxicity CK (U/l)
Phospholipasic A2 activity (U/mg)
Saline CA BthTX-I BthTX-I+CA BthTX-II BthTX-II+CA PrTX-I PrTX-I+CA PrTX-III PrTX-III+CA MjTX-II MjTX-II+CA
290.1 355.3 2200.4F133a 1303.7F128a b 3200.1F257a 1753.9F205a b 2253.6F132a 1030.3F164a b 2915.6F205a 1598.1F213a b 1913.4F161a 930.2F146a b
– 0.00 0.00 0.00 51.33 48.97 0.00 0.00 47.83 46.59 0.00 0.00
a b
Pb0.01 compared to the saline values. Pb0.01 compared to its PLA2 pair.
increase in the fluorescence signal (de Oliveira et al., 2001). Liposomes composed of a 9:1 molar ratio of egg yolk phosphatidylcholine (EYPC):dimyristoyl phosphatidic acid (DMPA) were prepared by reverse phase evaporation in a buffer (150 mM NaCl, 25 mM HEPES pH 7.0) containing 25 mM calcein (Sigma). The liposomes were passed through a 400-nm polycarbonate filter (Nucleopore, Pleasanton, CA, USA), and applied to a Sepharose 6 B column (18 cm) to separate the liposome entrapped calcein from the free calcein. Proteins and liposomes were mixed to a final toxin/lipid molar ratio of 1:200, and the kinetics of membrane damage was monitored by the increase in fluorescence emission at 520 nm with excitation at 490 nm following addition of the toxins. The fluorescence signal was expressed as the percentage of total calcein released following addition of 5 mM Triton X-100. The concentrations of toxins and CA were 3 and 4.5 Ag/ml, respectively, and Ca2+ was added to a final concentration of 2 mM and incubated for 30 min prior to assay.
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3. Results Table 1 presents the results obtained from the CK levels in mouse plasma and from the enzymatic activity assays measured using egg yolk as substrate. It is evident that there is no correlation between myotoxicity and phospholipase activity and that the presence of CA in the medium does not affect the hydrolytic activity of Asp49-PLA2s. The dose– effect curves for myotoxic and edema-forming activities are shown in Fig. 1. The myotoxic activity of PLA2s from the venom of Bothrops snakes promotes an increase of CK levels in mouse plasma. In the presence of CA, there was a significant decrease in the CK levels induced by PLA2s that indicates a 40% to 54% inhibition of the myotoxic effect. Bothrops phospholipases A2 also cause edema when injected into mouse paw (Fig. 2). CA significantly inhibited the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%) and PrTX-III (35%). In the case of
2.6. Fourier Transformed Infrared Spectroscopy analysis FT-IR Spectra were obtained on a Prote´ge´—460 MagnaIR Technology. BthTX-II and CA were previously incubated for 30 min and lyophilized before the assay. Approximately 100 Ag of the sample were mixed with 5 mg KBr (FT-IR grade) and analyzed along a 500–4000-cm1 range at a 4cm1 resolution. The computer program used to collect the data was Omnic and the results imported from Omnic to Origin 6. 2.7. Statistical analysis Results are presented as mean F standard error of mean (S.E.M.) of values obtained with the indicated number of animals. The statistical significance of differences between groups was evaluated using Student’s unpaired t-test. A P value b0.05 was considered to indicate significance.
Fig. 1. Representative dose-dependence inhibitory effect of crotapotin (CA) on myotoxic (A) and edema-inducing (B) activities of BthTX-II (*Pb0.01).
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Fig. 2. Effect of crotapotin (CA) on mouse paw edema induced by PLA2s isolated from Bothrops jararacussu BthTX-I (A) and BthTX-II (B), B. pirajai PrTX-I (C) and PrTX-III (D) and B. moojeni MjTX-II (E). The edema inducing response to crotapotin ( ) and PLA2s in the absence (E) or presence (z) of crotapotin is shown. The results represent the meanFS.E.M. of 12 animals. *Pb0.05, **Pb0.01 compared with the respective PLA2 (E).
!
MjTX-II, the 10% inhibitory effect reduced the edema to the levels observed in the controls used in this assay (saline and CA alone). The edema inducing experiment was performed over a 1 h period, the highest response appearing at 30 min. Sixty minutes after injection, the swollen paw was returning to normal. Possible secondary structural changes in the toxins on incubation with CA were evaluated with circular dichroism (CD) spectroscopy. The results for the mixture of PLA2 and
CA shown in Fig. 3 revealed that the sum of the two individual CD spectra of PrTX-I and CA were similar to the spectra of an equimolar mixture of the two proteins. This result indicates that the association between PrTX-I and CA does not change the secondary structure of either the toxin or the CA. Similar results were observed in the experiments using BthTX-I, BthTX-II and MjTX-II (data not shown). Amongst the Lys49-PLA2s tested, MjTX-II presented a significantly increased membrane damaging activity in
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Fig. 3. Far UV circular dichroism spectra of PrTX-I, CA and an equimolar mixture of both proteins. Spectra of PrTX-I (n) and CA ( ) alone, the mixture at a 1:1 molar ratio (4), and the calculated sum of the individual spectra for the PrTX-I and CA (E) are shown.
!
comparison with the other toxins, as evaluated by the release of entrapped calcein from liposomes (Fig. 4). In all cases, the membrane damaging activity of the toxins was reduced in the presence of CA, and although this reduction was slight, the effect was highly reproducible and statistically significant ( Pb0.05 in a paired t-test). FT-IR analysis showed that each protein has its own absorbance profile and that after complexation of CA with BthTX-II, the analysis reveals a third spectrum, suggesting an interaction among the structural groups at specific wavenumbers. In Fig. 5, the arrows show the presence of chemical groups in BthTX-II (Fig. 5A) and CA (Fig. 5B) that could interact with each other, whose signals decreased after complexation (Fig. 5C). Complexation of CA with other toxins showed similar spectra (data not shown). A band originating from COOH stretching vibrations, situated at 2856.77 cm1 for CA and 2931.33 for BthTX-II, refers to stretching vibrations of hydrocarbon aliphatic groups, e.g. CH2 and CH3 (Fig. 5A,B).
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necrosis and polymorphonuclear infiltration in the presence of CA 3 h after injection. This inhibitory action was most clearly observed when CA was incubated with MjTX-II, and in this case the morphological alterations were similar to the control (data not shown). Inhibition of edema by the association of CA with other PLA2s had been reported (Landucci et al., 2000). In our study, we observed a greater inhibition of the edema-forming activity when crotapotin was incubated with Asp49-PLA2s (30% inhibition) than with Lys49-PLA2s (13% inhibition), although we could not observe any significant inhibition of Asp49-PLA2 catalytic activity in the presence of CA. CA, however, inhibits the phospholipase activity of its natural complex, CB, and increases its lethality. It is known that crotoxin is formed by complexation of one molecule of CB with one molecule of CA, although free PLA2 can still be identified in the crude venom. Numerous natural inhibitors of toxic PLA2s have recently been isolated from the blood of a venomous snake and a marsupial (Soares et al., 1997, 2003) and from some plant extracts (Borges et al., 2000). Phospholipase inhibitors play an important role in the neutralization of neurotoxins and myotoxins, and some of these inhibitors may be useful in the future for envenomation treatment. Edema formation is a common feature of the inflammatory response and is dependent on a synergism between mediators that increase vascular permeability and those that increase blood flow (Williams and Morley, 1973; Williams and Peck, 1977). Histological analysis of the paw 30 min and 1 h after injection was performed (data not shown) and the pattern of the lesion showed a clear inflammation process with the absence of polymorphonuclear red blood cells and epidermal damage or necrosis. Since the paw returns to
4. Discussion Edema and myonecrosis are common effects of envenomation by snake bites, especially those of the genus Bothrops. The present study shows that a protein from the venom of a Crotalidae snake (C. d. terrificus), crotapotin (CA), is able to inhibit edema-inducing, myotoxic effects and liposome membrane disruption evoked by phospholipases A2 from the venom of several Bothrops snakes. In general, the combination of CA with PLA2 prior to injection resulted in significant reductions in both edema and myonecrosis. CA inhibited around 40–50% of the myotoxicity caused by PLA2s, as measured by plasma CK levels (Table 1). Histological analysis (data not shown) confirms the CK results, showing a decrease in muscle fiber
Fig. 4. Membrane damaging activities of the toxins (3 Ag/ml) alone and after incubation with CA (4.5 Ag/ml). The loss of liposome membrane integrity results in release of the fluorophore. The fluorescence signal was expressed as the percentage of total calcein released following addition of 5 mM Triton X-1000. *Pb0.05, compared with the respective PLA2.
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Fig. 5. Fourier Transform Infrared Spectroscopy (FT-IR) was obtained for BthTX-II (A), crotapotin (B) and the complex BthTX-II+CA (C). The arrows show the modifications observed on both spectra after formation of the complex. Note that these modifications are evidenced by alterations in the wavenumber parameters.
normal after 3 h, we suggest that the inflammation process is restricted to the area of injection. The presence of aspartic acid at position 49 is crucial for calcium binding to the Asp49-PLA2s and Ca2+ is essential for catalytic activity (Scott et al., 1992). Despite the lack of an aspartic acid residue at position 49, the Lys49-PLA2 proteins are potent inducers of myonecrosis by one or more unknown mechanisms (Homsi-Brabdeburgo et al., 1988; Johnson and Ownby, 1993). Selistre de Arau´jo et al. (1996a) observed that Lys49-PLA2s comprise a highly conserved protein family very distinct from the Asp49-PLA2 protein family, and comparison of Lys49-PLA2 amino acid sequences reveals a high level (70–95%) of identity. In contrast, amino acid identity within the Asp49 group can be as low as 47% in the case of the Asp49-PLA2 from Crotalus adamanteus (Selistre de Arau´jo et al., 1996b). Despite these differences in the degree of amino acid conservation within the Asp49-PLA2s and the Lys49-PLA2s, the results reported here show that there are interactions between crotapotin and both Asp49- and Lys49-PLA2s, from the venoms of different species.
Although no indication of structural modifications was detectable by either CD or FT-IR analysis, an alteration in absorbance at wavenumber=2856 cm1 in the FT-IR spectrum for the carboxyl terminal groups of crotapotin suggests an ionic interaction, and similar alterations for the aliphatic carbon skeleton indicate hydrophobic interactions at wavenumber 2931 cm1 for BthTX-II and 2924 cm1 for crotapotin (Fig. 5). The differences observed in Fig. 5C are in the range 2873.40–3064.63 cm1, where those groups should vibrate. There is a clear decrease of the stretching vibration, suggesting a steric impeachment for those groups to move freely. This may be explained by interactions with other PLA2s, forming homodimers (da Silva Giotto et al., 1998). It has been reported that CA is a PLA2-like protein (Bouchier et al., 1991), so it is likely to interact with other PLA2s as it interacts with the CB in the C. d. terrificus venom to form the neurotoxin crotoxin. Analysis of infrared spectra allows us to state that there are interactions between CA and BthTX-II that involve carboxyl and aliphatic groups. The nature of the biochemical interactions between PLA2s and crotapotin remains to be established and may
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involve chemical impairment of some amino acid residues to properly play their functional role (Soares et al., 2001). In the liposome membrane disruption assay, the MjTX-II was shown to have increased activity when compared with other PLA2s tested. This may be a consequence of a peculiar amino acid sequence in the C-terminal loop region of this protein. Recent site-directed mutagenesis results with BthTX-I have demonstrated that alterations in the physical–chemical properties of this region have significant effects on the membrane damaging effect (Chioato et al., 2002). In this report, we have presented evidence that crotapotin inhibits the edema and myotoxic activities of several Bothrops PLA2, and this effect could be related to the prevention of membrane disorganization since liberation of calcein in the liposome assay was shown to be significantly decreased when PLA2 was incubated with CA. As our data point to blockage of strategic binding sites of BthTX-II by CA, rather than to detectable modifications of its secondary structure, it is plausible to assume that inhibition of liposome disruption is a consequence of a partial hindrance of binding to its membrane surface due to the above mentioned blockage, which would be equivalent to sitedirected mutagenesis of specific amino acids involved with the binding event (Chioato et al., 2002). Inhibition of PLA2 toxicity without inducing structural alterations would be required to definitively specify the myotoxic action of Lys49-PLA2s and Asp49-PLA2s. Previous experiments suggest that both hydrolytic activity and biological effects do not occur in the same catalytic site (Soares et al., 2001). Crotapotin displays a significant inhibitory effect on Bothrops PLA2s, oppositely to what happens with its natural Crotalus PLA2 counterpart, which is strongly potentiated by this acidic protein. Although CA potentiates CB at a 1:1 ratio (mol/ mol), excess CA inhibits its edema inducing, neurotoxic and anti-bacterial activities (Cecchini et al., 2004). Not surprisingly, C. d. terrificus venom displays excess PLA2s over CA, although these PLA2s are apparently CB homologous (work in preparation). More research is still necessary to fulfill the requirements for use of crotapotin for therapeutics of envenomation by Bothrops venoms or other PLA2-containing venoms. In addition, this work points to the use of CA as a promising tool to explore PLA2s structure and its relationship to biological activity. Acknowledgements The authors gratefully acknowledge the financial support from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), as well as the skillful technical assistance of Odete A.B. Cunha and Carlos A. Vieira.
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