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Chemistry » Analytical Chemistry » "Chromatography - The Most Versatile Method of Chemical Analysis", book edited by Leonardo de Azevedo Calderon, ISBN 978-953-51-0813-9, Published: October 24, 2012 under CC BY 3.0 license. © The Author(s).

Chapter 1

Purification of Phospholipases A2 from American Snake Venoms

By Rodrigo G. Stábeli, Rodrigo Simões-Silva, Anderson M. Kayano, Gizeli S. Gimenez, Andrea A. Moura, Cleópatra A. S. Caldeira, Antonio Coutinho-Neto, Kayena D. Zaqueo, Juliana P. Zuliani, Leonardo A. Calderon and Andreimar M. Soares
DOI: 10.5772/53052

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Phospholipase hydrolysis specificity sites in a 1,2-diacylglycerolphospholipid molecule (structure design from the ACD/l Lab. via Chem. Sketch – Freeware Version 1994 – 2009 software).
Figure 1. Phospholipase hydrolysis specificity sites in a 1,2-diacylglycerolphospholipid molecule (structure design from the ACD/l Lab. via Chem. Sketch – Freeware Version 1994 – 2009 software).
Schematic representation of the catalysis mechanism proposed for the PLA2s. Interaction of the residues from the catalytic site of sPLA2s and the calcium ion with the transition state of the catalytic reaction in which a water molecule polarized by the His48 and Asp99 residues binds to the carbonyl group of the substrate [18].
Figure 2. Schematic representation of the catalysis mechanism proposed for the PLA2s. Interaction of the residues from the catalytic site of sPLA2s and the calcium ion with the transition state of the catalytic reaction in which a water molecule polarized by the His48 and Asp99 residues binds to the carbonyl group of the substrate [18].
Chromatographic profile using CM-sepharose® Column 1ml (Hitrap) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1 M (buffer B) in pH 8.0.A. Chromatography of the crude venom from Bothrops brazili B. Chromatography of the crude venom from Bothrops moojeni C. Chromatography of the crude venom from Bothrops jararacussu. Absorbance read at 280 nm.,2,3,4,5 and 6 marks indicate the fractions corresponding to the PLA2s of each venom.
Figure 3. Chromatographic profile using CM-sepharose® Column 1ml (Hitrap) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1 M (buffer B) in pH 8.0.A. Chromatography of the crude venom from Bothrops brazili B. Chromatography of the crude venom from Bothrops moojeni C. Chromatography of the crude venom from Bothrops jararacussu. Absorbance read at 280 nm.,2,3,4,5 and 6 marks indicate the fractions corresponding to the PLA2s of each venom.
Chromatographic profile of the B.jararacussu venom in CM-sepharose® column 1 ml (Hitrap) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1M (buffer B) in different pH conditions. A. pH 5.0 B. pH 6.0 C. pH 7.0 D. pH 8.0. Absorbance was read at 280 nm. Fractions numbered (1 to 8) indicate the fractions selected for SDS-PAGE analysis in order to confirm the presence of PLA2s (BthTx I e BthTx II).
Figure 4. Chromatographic profile of the B.jararacussu venom in CM-sepharose® column 1 ml (Hitrap) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1M (buffer B) in different pH conditions. A. pH 5.0 B. pH 6.0 C. pH 7.0 D. pH 8.0. Absorbance was read at 280 nm. Fractions numbered (1 to 8) indicate the fractions selected for SDS-PAGE analysis in order to confirm the presence of PLA2s (BthTx I e BthTx II).
SDS Page analysis. Lines 1 and 2 (pH 5.0); 3 and 4 (pH 6.0); 5 and 6 (pH 7.0); 7 and 8 (pH 8.0). BthTx I was obtained in high degree of purity with pHs 5.0, 6.0, and 8.0. BthTx II was obtained with pH 7.0.
Figure 5. SDS Page analysis. Lines 1 and 2 (pH 5.0); 3 and 4 (pH 6.0); 5 and 6 (pH 7.0); 7 and 8 (pH 8.0). BthTx I was obtained in high degree of purity with pHs 5.0, 6.0, and 8.0. BthTx II was obtained with pH 7.0.
Electrophoretic profile in 2D-PAGE 10%, 13 cm strip pH 3-10 non-linear of proteins from crude venom from Bothrops moojeni. Molecular weight (MW) –Color Plus Prestained Protein Marker – Broad Range (7-175 kDa) (P7709S). Coomassie G-250.
Figure 6. Electrophoretic profile in 2D-PAGE 10%, 13 cm strip pH 3-10 non-linear of proteins from crude venom from Bothrops moojeni. Molecular weight (MW) –Color Plus Prestained Protein Marker – Broad Range (7-175 kDa) (P7709S). Coomassie G-250.

Purification of Phospholipases A2 from American Snake Venoms

Leonardo de A. Calderon, Juliana P. Zuliani1, Rodrigo G. Stábeli1, Andreimar M. Soares2, Anderson M. Kayano3, Antonio C. Neto3, Andrea A. de Moura3, Gizeli S. Gimenez3, Kayena D. Zaqueo4, 5, Rodrigo S. Silva4, 5 and Cleópatra A. S. Caldeira6

1. Introduction

Snake venoms are a complex mixture of compounds with a wide range of biological and pharmacological activities, which more than 90% of their dry weight is composed by proteins, comprising a variety of enzymes, such as proteases (metalo and serine), phospholipases A2, L-aminoacid oxidases, esterases, and others [1-5]. A great number of proteins were purified and characterized from snake venoms [1, 2]. Some of these proteins exhibit enzymatic activity, while many others are non-enzymatic proteins and peptides. Based on their structures, they can be grouped into a small number of super-families based on remarkable similarities in their primary, secondary and tertiary structures, however showing distinct pharmacologic effects [3].

One of the most important protein super-families present in snake venoms are the phospholipases A2 (PLA2, E.C., a class of heat-stable and highly homologous enzymes, which catalyse the hydrolysis of the 2-acyl bond of cell membrane phospholipids releasing arachidonic acid and lysophospholipids (Figure 1). These proteins are found in a wide range of cells, tissues and biological fluids, such as macrophages, platelets, spleen, smooth muscle, placenta, synovial fluid, inflammatory exudate and animal venoms. There is a high medical and scientific interest in these enzymes due to their involvement in a variety of inflammatory diseases and accidents caused by venomous animals. Since the first PLA2 activity was observed in Naja snake venom, PLA2s were characterized as the major component of snake venoms, being responsible for several pathophysiological effects caused by snake envenomation, such as neurotoxic, cardiotoxic, myotoxic, cytotoxic, hypotensive and anti-coagulant activities [1-10].

Phospholipases constitute a diverse subgroup of lipolytic enzymes that share the ability to hydrolyse one or more ester linkages in phospholipids, with phosphodiesterase as well as acyl hydrolase activity. The amphipathic nature of phospholipids creates obstacles for the enzymes, as the substrates are assembled into bilayers or micelles and are not present in significant amounts as a single soluble substrate [11]. According to Waite [12], all phospholipases target phospholipids as substrates, they vary in the site of action on the phospholipid molecule, their function and mode of action, and their regulation. Phospholipases function in various roles, ranging from the digestion of nutrients to the formation of bioactive molecules. This diversity of function suggests that phospholipases are relevant for life; the continuous remodelling of cell membranes requires the action of one or more phospholipases. The most common phospholipids in mammalian cells are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). The plasma membrane of most eukaryotic cells contains predominantly PC and sphingomyelin in the outer leaflet, and PI, PE and PS in the inner leaflet [11].


Figure 1.

Phospholipase hydrolysis specificity sites in a 1,2-diacylglycerolphospholipid molecule (structure design from the ACD/l Lab. via Chem. Sketch – Freeware Version 1994 – 2009 software).

Phospholipases are classified according to their site of action in the phospholipid molecule. Thus, a phospholipase A1 (PLA1) hydrolyzes the 1-acyl group of a phospholipid, the bond between the fatty acid and the glycerine residue at the 1-position of the phospholipid. A phospholipase A2 (PLA2) hydrolyzes the 2-acyl, or central acyl, group and phospholipases C (PLC) and D (PLD), which are also known as phosphodiesterases, cleave on different sides of the phosphodiester linkage (Figure 1). The hydrolysis of a phospholipid by a PLA1 or a PLA2 results in the production of a lysophospholipid. The phospholipase metabolites are involved in diverse cellular processes including signal transduction, host defense (including antibacterial effects), formation of platelet activating cofactor, membrane remodeling and general lipid metabolism [12-14].

According to the latest classification [6], these proteins constitute a superfamily of different enzymes belonging to 15 groups and their subgroups including five distinct types of enzymes: the ones called secreted PLA2 (sPLA2), the cytosolic (cPLA2), the Ca2+ independent (iPLA2), the acetyl-hydrolases from platelet activating factors (PAF-AH) and the liposomal. The classification system groups these enzymes considering characteristics such as their origin, aminoacid sequence and catalytic mechanisms, among others.

The sPLA2s have a Mr. varying from 13,000 to 18,000, usually containing from 5 to 8 disulphide bond. They are enzymes that have a histidine in the active site and require the presence of the Ca2+ ion for the catalysis. Phospholipases A2 from the IA, IB, IIA, IIB, IIC, IID, IIE, IIF, III, V, IX, X, XIA, XIB, XII, XIII, XIV groups are representative of the sPLA2s. The cPLA2s are proteins with Mr between 61,000 to 114,000 that also use a serine in the catalytic site (groups IVA, IVB, IVC, IVD, IVE, IVF). The iPLA2s are enzymes which also use a serine for catalysis (groups VIA-1, VIA-2, VIB, VIC, VID, VIE, VIF). The PAF-AH are phospholipases A2 with serine in the catalytic site that hydrolyze the acetyl group from the sn-2 position of the platelet activating factors (PAF), whose representative groups are VIIA, VIIB, VIIIA, VIIB. The liposomal PLA2s are assembled in group XV and are enzymes with an optimum pH close to 4.5 that have preserved histidine and aspartate residues, suggesting the presence of the catalytic triad Ser/His/Asp and also a supposed sequence N-terminal sign and N-bond glycosylation sites [6].

With the discovery of a great variety of phospholipase A2 in the last decade and the present expansion of the research in the area, more PLA2s should be discovered yet. Phospholipase A2 found in snake venoms (svPLA2s) are classified into groups I and II. The phospholipase A2 from group I have two to three amino acids inserted in the 52-65 regions, called “elapid loop”, being isolated from the snake venoms of the Elapidae family (subfamily: Elapinae and Hydrophiinae). The ones from group II are characterized by the lack of the Cys11-Cys77 bond which is substituted by a disulphide bond between the Cys51-Cys133, and besides that had five to seven amino acids extending the C-terminal regions, being bound in snake venoms of the Viperidae family (subfamily Viperinae and Crotalinae) [15,16].

The myotoxic PLA2s of the IIA class have been subdivided in two main groups: The Asp49, catalytically active; and the Lys49, catalytically inactive. The essential co-factor for the phospholipase A2 catalysis Ca2+. The phospholipase A2 Asp49 require calcium to stabilize the catalytic conformation, presenting a calcium bond site that is constituted by the β-carboxylic group of Asp49 and the C=O carbonylic groups of the Tyr28, Gly30 and Gly32. The presence of two water molecules structurally preserved complete the coordination sphere of Ca2+ forming a pentagonal pyramid [9,15].

The catalytic mechanism of the PLA2-phospholipid involves the nucleophilic attack of a water molecule to the sn-2 bond of the phospholipid substrate (Figure 2). In the proposed model, the proton from position 3 of the imidazole ring of the His48 residue involved in a strong interaction with the carboxylate group of the Asp49 prevents the imidazole ring rotation to occur and keeps the nitrogen at position 1 of this ring, in an appropriate special position. A water molecule then promotes the nucleophilic attack to the carbon of the ester group of the substrate and, at this moment, the imidazole ring of the His48 receives a proton from the water molecule, favoring the reaction. Subsequently to the acyl-ester bond hydrolysis at the sn-2 position of the phospholipid, this proton is donated by the imidazole ring to the oxygen, which then forms the alcohol group of the lysophospholipid to be released together with the fatty acid [15,17].

The Ca2+ ion, coordinated by the Asp49 residue, a water molecule and the oxygen atoms from the Gly30, Trp31 and Gly32 (not shown), are responsible for the stabilization of the reactive intermediary [15].


Figure 2.

Schematic representation of the catalysis mechanism proposed for the PLA2s. Interaction of the residues from the catalytic site of sPLA2s and the calcium ion with the transition state of the catalytic reaction in which a water molecule polarized by the His48 and Asp99 residues binds to the carbonyl group of the substrate [18].

The substitution of the Asp49 residue by the Lys49 significantly alters the binding site of Ca2+ in the phospholipase A2, preventing its binding and resulting in low or inexistent catalytic activity. Thus, the Asp49 residue is of fundamental importance for the catalytic mechanism of the phospholipase A2. It is likely that this occurs due to its capability of binding and orienting the calcium ion, however, there is no relevant difference between Asp49 and Lys49 in relation to the structural conformation stability of these enzymes [9,15,19].

The absence of catalytic activity does not affect myotoxicity. Most snake PLA2s from the Bothrops genus already described are basic proteins, with isoelectric point between 7 to 10, showing the presence or absence of catalytic, myotoxic, edematogenic and anticoagulating activities [9,20].

On the other hand, acid PLA2s present in Bothrops snake venoms were not studied as well as basic PLA2s, resulting in little knowledge regarding the action mechanism of these enzymes [21-25].

PLA2s catalytic activity represents a key role in envenomation pathophysiology, however, recent studies have shown that some effects are independent of PLA2s catalytic activity, such as myotoxicity [19,26]. The absence of a tight correlation between PLA2 catalytic and non-catalytic activities, together with the diversity of biological effects produced by these proteins increases the scientific interest in the understanding of the structural basis of PLA2 mechanisms of action.

Evidences suggest that these activities can be mediated by interactions between PLA2s and endogen acceptors on the target cell membrane [27-29].

2. PLA2 purification

Snake venom components, obtained with high degree of purity, could be used for the understanding of the role of these components in the physiopathological processes resulted from poisoning, as well as biotechnological/nanotechnological applications. Hence, many purified PLA2s from snake venoms, as well as epitopes of these molecules, are being mapped in order to identify determinants responsible for the deleterious actions seen, as well as possible applications in biotechnological models.

New advances in materials and equipments have contributed with protein purification processes, allowing the obtaining of samples with high degree of purity and quantity. These advances have allowed process optimization, providing reduction of steps, reagents use and thus avoiding the unnecessary exposure to agents that may, in some way, alter the sample’s functionality or physical-chemical stability.

Thus, the selection of adequate techniques and chromatographic methods oriented by physical chemical properties and biological/functional characteristics, are of fundamental importance to obtain satisfactory results. The information pertinent to protein structure, such as the homology to others already purified, should be taken into consideration and could make the purification processes easier.

Ion exchange chromatography was introduced in 1930 [30] and still one of the main techniques used for protein purification. It has been extensively used in single step processes as well as associated to other chromatographic techniques. Ion exchange chromatography allows the separation of proteins based on their charge due to amino acid composition that are ionized as a function of pH.

Proteins with positive net charge, in a certain pH (bellow their isoelectric point), can be separated with the use of a cation exchange resin and on the other hand, proteins with negative net charge in a pH value above their isoelectric point, can be separated with an anion exchange resin.

Scientific publications have shown that the use of cation-exchange resins is a very efficient method to obtain PLA2s from bothropic venoms, particularly those with alkaline pH (Table 1). The versatility of this technique can be observed in the work done by Andriao-Escarso et al. [21] who compared the fractioning of many bothropic venoms. In this work, the venoms were fractioned in a column containing CM-Sepharose® (2 x 20 cm), equilibrated with ammonium bicarbonate 50 mM pH 8.0 and eluted with a saline gradient of 50 to 500 mM of the same reagent. Under these conditions, MjTX-I and MjTX-II from B. moojeni snake venom were co-purified (isoforms of PLA2 with pIs of 8.1 and 8.2 values, respectively). The same occurs with B. jararacussu venom, where the BthTX-I and BthTX-II were purified. However, the most expressive result was observed with B. pirajai venom, from which 3 isoforms of myotoxins, called as PrTX-I (pI 8.50), PrTX-II (pI 9.03) and PrTX-III (pI 9.16) were purified. In the above cases, it is important to note that the protein elution occurs always following pIs increasing value. In our lab we used this technique routinely in order to isolate myotoxins from bothropic venoms, which can be observed in the chromatograms shown in Figure 3.


Figure 3.

Chromatographic profile using CM-sepharose® Column 1ml (Hitrap) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1 M (buffer B) in pH 8.0.A. Chromatography of the crude venom from Bothrops brazili B. Chromatography of the crude venom from Bothrops moojeni C. Chromatography of the crude venom from Bothrops jararacussu. Absorbance read at 280 nm.,2,3,4,5 and 6 marks indicate the fractions corresponding to the PLA2s of each venom.


Table 1.

PLA2s isolated from American snake venoms and respective chromatographic methods used.

Some authors have proposed changes to the methodology described above. Spencer et al. [31] described the purification of BthTX-I with the use of Resourse S® (methyl-sulphonate functional group), equilibrated in pH 7.8 (phosphate buffer 25 mM). Sample elution was done in increasing ionic strength conditions (NaCl 0 to 2 M), under 2.5 ml/min flow. In this model, the BthTX-I was eluted in NaCl 0.42M with a high degree of purity. However, the chromatographic profile in the conditions tested differs significantly from the observed in other works that describe the fractioning of this venom. This difference is due to the resin composition. This is corroborated with data obtained in experiments performed in our lab, where the effect of pH in the separation of myotoxin isoforms from B. jararacussu venom was used, as shown in Figures 4. SDS-PAGE showed that fractions corresponding to myotoxins showed protein bands with apparent molecular mass compatible with PLA2s class II (Figure 5).


Figure 4.

Chromatographic profile of the B.jararacussu venom in CM-sepharose® column 1 ml (Hitrap) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1M (buffer B) in different pH conditions. A. pH 5.0 B. pH 6.0 C. pH 7.0 D. pH 8.0. Absorbance was read at 280 nm. Fractions numbered (1 to 8) indicate the fractions selected for SDS-PAGE analysis in order to confirm the presence of PLA2s (BthTx I e BthTx II).


Figure 5.

SDS Page analysis. Lines 1 and 2 (pH 5.0); 3 and 4 (pH 6.0); 5 and 6 (pH 7.0); 7 and 8 (pH 8.0). BthTx I was obtained in high degree of purity with pHs 5.0, 6.0, and 8.0. BthTx II was obtained with pH 7.0.

Resolution differences were also observed by other authors. As performed by Lomonte et al. [26], the isolation of two basic myotoxins, MjTX-I e MjTX-II, from the B. moojeni venom was obtained using CM-Sephadex C-25 equilibrated with Tris-HCl 50 mM pH 7.0 and eluted in saline gradient up to 0.75 M of Tris-HCl. Also, Soares et al. [33] described the isolation of MjTX-II with high purity using the combination of CM-Sepharose resin and ammonium bicarbonate buffer. According to the authors, the increase of pH to 8.0 has favored the elution of several fractions, allowing MjTX-II to be eluted separately with ionic strength equal to 0.35 M of ammonium bicarbonate. Moreover, the use of CM-Sepharose® seems to have also contributed a lot in the increasing of resolution for this chromatographic separation.

The combination of chromatographic techniques has also been used to purify these toxins. The association of the Ion-exchange chromatography and molecular exclusion has been one of the most recurrent in isolation and purification of phospholipases from bothropic venoms. Gel filtration chromatography is a technique based in particle size to obtain the separation. In this type of separation there is no physical or chemical interaction between the molecules of the analyte and the stationary phase, being currently used for separation of molecules with high molecular mass. The sample is introduced in a column, filled with a matrix constituted by small sized silica particles (5 to 10 µm) or a polymer containing a uniform net pores of which solvent and solute molecules diffuse. The retention time in the column depends on the effective size of the analyte molecules, the higher sized being the first ones to be eluted. Different from the higher molecules, the smaller penetrate the pores being retained and eluted later. Between the higher and lower molecules, there are the intermediary sized molecules, whose penetration capacity in the pores depends on their diameter. In addition to that, this technique has also some very important characteristics, such as operational simplicity, physical chemical stability, inertia (absence of reactivity and adsorptive properties) and versatility, since it allows the separation of small molecules with mass under 100 Da as well as extremely big molecules with various kDa.

The work performed by Homsi-Brandeburgo et al. [34] is a example of combination of different chromatographic techniques for the isolation of myotoxins with PLA2 structure. It describes for the first time the BthTX-I purification using the combination of molecular exclusion chromatography in Sephadex G-75® resin followed by Ionic exchange chromatography in SP-Sephadex C-25®. In the first step, four fractions were obtained, called SI, SII, SIII and SIV. The Functional analysis of these fractions showed that the proteolytic activity over casein and fibrinogen was detected on fraction SI, while the phospholipase activity was concentrated in fraction SIII. The apparent molecular mass profile of this fraction showed that it was composed by proteins between 12,900 and 28,800 Da, compatible with the mass profile of the class II PLA2s.

On the second step, SIII fraction was submitted to ionic exchange chromatography and five fractions were obtained, identified as SIIISPI to SIIISPIV. The pIs and apparent molecular mass evaluation showed the following profile: SIIIPI (pI 4.2 and 22.400 Da), SIIIPII (pI 4.8 and 15.500 Da), SIIIPIII (pI 6.9 and dimeric structure, each monomer with a molecular mass of 13.900 Da), SIIIPIV (pI 7.7 and 13.200 Da) e SIIIPV called BthTX-I that presented pI 8,2 and 12.880 Da. Pereira et al. [35] obtained the complete sequence of BthTX-II, a myotoxin homologous to the BthTX-I, which corresponds to the SIIISPIV fraction described by Homsi-Brandeburgo et al. [34].

Another chromatographic technique regularly used in PLA2s purification procedures is the Reverse-phase associated with High performance liquid chromatography (RP- HPLC). This technique is characterized by its high resolution capacity and is normally used in a more refined step of the purification process, being very useful in separating isoforms. The retention principle of reverse-phase chromatography is based in hydrophobicity and is mainly due to the interactions between hydrophobic domains of the proteins and the stationary phase. This technique has many advantages, such as: use of less toxic mobile phases together with lower costs, such as methanol and water; stable stationary phases; fast column equilibrium after mobile phase change; easy to use gradient elution; faster analysis and good reproducibility.

Rodrigues et al. [36] described the isolation of two PLA2s isoforms from the B. neuwiedi pauloensis venom using the combination of ion (cation) exchange chromatography and molecular exclusion setting up a preparative phase. Subsequently, a reverse-phase chromatography was used for the analytical phase of the procedure. Initially, the venom was fractioned in a column containing CM-Sepharose® equilibrated with ammonium acetate solution 0.05 M, pH 5.5 and eluted in linear gradient up to 1 M of the same buffer, resulting in six fractions. The pH, more acid than the ones used in the work previously mentioned, has increased the surface residual charge, intensifying the interaction force between the protein and the resin, thus altering the elution profile when compared to the performed by Rodrigues et al. [37]. Proceeding with purification, the sample with phospholipase activity (S-5) was submitted to a new fractioning in a Sephadex G-50® column yielding 3 fractions, of which the denominated S-5-SG-2 showed catalytic activity. It was then submitted to RP- HPLC in C18 column to obtain toxins with high purity degree.

Also, with the use of a multiple step procedure [38] successfully isolated two isoforms of PLA2s from B. leucurus venom. After a first molecular exclusion chromatography using Sephacryl S-200®, 7 fractions were obtained, from which the named “P6” showed to be composed by proteins with apparent molecular mass bellow 30 kDa, and a major fraction of approximately 14 kDa concentrated the phospholipase activity. This fraction was re-chromatographed in a Q-Sepharose® resin (ion exchange) and equilibrated with Tris-HCl 20 mM pH 8.0, yielding 6 fractions. The fraction corresponding to the negatively charged fraction was eluted without significant interaction with the resin, hence with a positive residual charge (basic pI) was selected, showing to be a homogeneous fraction of 14 kDa and presenting phospholipase activity. This fraction was submitted to a RP- HPLC in C4 column, yielding as result two major fractions with close hydrophobicity (eluted with 33% and 36% acetonitrile) and apparent molecular mass of 14 kDa.

Myotoxins with PLA2s structure from bothropic venoms that have acid pI have being more difficult to isolate. Different from cation exchange resins (CM Sepharose®, Resource S® and CM Sephadex®), anion exchange resins have not been so efficient in the separation of components from bothropic venoms, which requires, complementary steps to obtain these toxins with a satisfactory purity degree, as shown in Table 1.

Daniele et al. [32] described the fractioning of the B. neuwiedii venom using a combination of double molecular exclusion chromatography followed by anion exchange chromatography. The first step of the molecular exclusion chromatography was done using Sephadex G-50® where a single fraction with PLA2s activity was eluted. This fraction was re-chromatographed in Sephacryl S-200® resin, yielding 2 active fractions. The first fraction was re-chromatographed in Mono Q® column (functional group quaternary ammonium) yielding a PLA2s named P-3. From the second fraction, submitted to the same chromatographic procedure, two other PLA2s isoforms were isolated, named P-1 and P-2. Although showing different behavior over the molecular exclusion resin, the three isoforms showed very close apparent molecular mass (15 kDa) when assayed by SDS-PAGE. This difference could be resulted from differential interactions of aromatic residues located on the protein surface with the stationary phase [40, 41] and can be also verified in other acid PLA2s, like the one obtained from B. jararacussu venom by Homsi-Brandeburgo et al. [34].

Other procedures used hydrophobic interaction chromatography to isolate these PLA2s. This is a method that separates the proteins by means of their hydrophobicity: the hydrophobic domains of the proteins bind to the hydrophobic functional groups (phenyl and aryl) of the stationary phase. Proteins should be submitted to the presence of a high saline concentration, which stabilize then and increases water entropy, thus amplifying hydrophobic interactions. In the presence of high salt concentrations, the matrix functional groups interact and retain the proteins that have surface hydrophobic domains. Hence, elution and protein separations can be controlled altering the salt or reducing its concentration.

Santos-Filho et al. [42], working with B. moojeni venom, applied three sequential steps to obtain BmooTX-I, a PLA2 with apparent molecular mass of 15 kDa and pl 4.2. In this work, the crude venom was chromatographed in DEAE-Sepharose® (Dietylaminoetyl) resin, equilibrated with ammonium bicarbonate 50mM, pH 7.8 and brought to a saline gradient of 0.3M of the same salt. A fraction named E3 showed phospholipase activity, being then submitted to molecular exclusion chromatography in Sephadex G-75® resin. Three fractions were obtained, from which one named S2G3 was submitted to hydrophobic interaction chromatography in Phenyl-Sepharose® resin, the BmooTX-I being eluted in the end of the process.

In a work published in 2011, Nunes et al. [43] described the isolation of an acid phospholipase named BL-PLA2, obtained from Bothrops leucurus through two sequential chromatographic steps. On the first step, the acid proteins were separated from the others with the use of a cation exchange column (CM-Sepharose®) equilibrated with ammonium bicarbonate, pH 7.8. The acid fraction (eluted without interaction with the resin) was lyophilized and applied to a Phenyl-Sepharose CL-4B® column (1 x 10 cm), previously equilibrated with a Tris-HCl 10mM buffer, NaCl 4M, pH 8.5. The elution occurred under decreasing NaCl gradient in a buffered environment (Tris-HCl 10 mM, pH 8.5), concluding the process in an electrolyte free environment. An enzymatically active fraction (BL-PLA2), (with pI 5.4 and apparent molecular mass of approximately 15 kDa) was obtained at the end of the process.

The bioaffinity chromatography differs from others chromatographic methods because it is based in biological or functional interactions between the protein and the ligand. The nature of these interactions varies, being the most used those which are based on the interactions between: enzymes and substrate analogous and inhibitors; antigens and antibodies; lectins and glycoconjugates; metals and proteins fused with histamine tails. The high selectivity, the easiness of performance together with the diversity of ligands that can be immobilized in a chromatographic matrix make this method a useful tool for the purification of phospholipases. Based on the neutralization of myotoxic effects of the venom from B. jararacussu by heparin [44-46], the use of a column containing Agarose-heparin® could be used for the purification of myotoxins. They also ratify the interactions between heparin and myotoxin through the reduction of many biological effects, such as: edema induction, myotoxicity (in vivo) and cytotoxicity over mice myoblasts culture (L.6 – ATCC CRL 14581) and endothelial cells.

Following this strategy, Soares et al. [26] described the purification of BnSP-7, a myotoxin Lys-49 from B. neuwiedi pauloensis, with the use of chromatographic process based in this heparin functionality, which corroborates previous results obtained by Lomonte et al. [46], that showed the efficient inhibitory activity of heparin against myotoxicity and edema induced by myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper. Also in this study, it was possible to infer the participation of the C-terminal region of the protein in the damaging effects on the cytoplasmic membrane.

Snake venom components share many similar antigenic epitopes that can induce to a crossed recognition by antibodies produces against a determined toxin. In this context, Stabeli et al. [47] showed that antibodies that recognize a peptide (Ile1-Hse11) from Bm-LAAO present crossed immunoreactivity with components not related to the LAAOs group present in venoms from Bothrops, Crotalus, Micrurus e Lachesis snake venoms. Also, Beghini et al. [48] showed that the serum produced against crotoxin and phospholipase A2 from Crotalus durissus cascavella was able to neutralize the neurotoxic activity produced by B. jararacussu venom and BthTX-I.

Based on this information, pertinent to the crossed immunoreactivity existent between venom components, Gomes et al. [49] described the co-purification of a lectin (BJcuL) and a phospholipase A2 (BthTX-1) using a immunoaffinity resin containing antibodies produced against the crotoxin. 20 mg of crotoxin was solubilized in coupling buffer (sodium bicarbonate 100 mM, NaCl mM, pH 8.3) and incubated overnight at 4 °C with 1 g of Sepharose® activated by cyanogen bromide (CNBr). After washing with the same buffer, the resin was blocked with Tris-HCl 100 mM buffer. This resin was packed and thoroughly washed with saline phosphate buffer (PBS) pH 7.4. Crotalic counter-venom hiperimune horse plasma (20 mg) was applied over the resin at a flow of 10 mL/hr and re-circulated overnight through the column. Then, it was washed until the absorbance went back to basal levels, showing that the material was retained (IgG anti-Ctx), then eluted with glycin-HCl 100 mM pH 2.8. The IgG anti-Ctx was then immobilized in CNBr activated Sepharose® resin through a procedure analogous to the above cited, generating a new resin called Sepharose-Bound Anti-CtxIgG. 20 mg of the crude venom from B. jararacussu was applied over this resin, yielding two fractions: the first, composed by proteins that were not recognized by the immobilized antibodies and a second fraction composed by components of venom from B. jararacussu that reacted crosswise with the Anti-Ctx antibodies, called Bj-F. A posterior analysis of this fraction, done by mass spectrometry, amino-terminal sequencing by Edman degradation and search by homology in the NCBI protein data bank, showed that it was composed by lectin and BthTX-I.

Different authors used substrate analogous or reversible inhibitors coupled to the chromatographic resin. Rock and Snyder [50] were the first ones to use phospholipid analogous to build a bioaffinity matrix [Rac-1-(9-carboxy)-nonil-2-exadecilglycero-3-phosphocoline]. In addition to them, Dijkman [51] described the synthesis of an analogous of acylamino phospholipid[(R)-1-deoxy-1-thio-(ω-carboxy-undecyl)-2-deoxy- (n-decanoylamino)-3-glycerophosphocholine] which was coupled to a Sepharose 6B® resin containing a spacer arm. With the use of this resin it was possible to purify phospholipases from horse pancreas, and venoms from Naja melanonleuca and Crotallus adamanteus.

3. Characterization

Venomic can be defined as an analysis in large scale of the components present in the venom of a certain species. In this context, the proteomic approach has allowed a better understanding of the venom components, through the application of many instruments that enables the analysis of their expression, structure, pos-traductional modifications and classification by homology or function. An approach developed by Calvete [52] for the analysis of snake venomic consists in an initial fractioning step of the crude venom using RP - HPLC, followed by characterization of each fraction by a combination of amino-terminal sequencing, SDS-PAGE, IEF or 2DE and mass spectrometry to determine molecular mass and cysteine content. Additionally, the modern venomic analysis use techniques such as Peptide Mass Fingerprint and the search for sequence similarity in data banks.

SDS-PAGE is a method related to the migration of charged particles in a medium under the influence of a continuous electric field [53]. From the electrophoretic point of view, the most important properties of the proteins are molar mass, charge and conformation. Mono dimensional polyacrylamide gel electrophoresis permit the analysis of the protein in its native or denatured form. In the first case, there are no alterations in conformation, biological activity and between protein subunits. This system is called non-dissociating or native, which proteins are separated based on their charge, using the isoelectric focusing method (IEF), or else, in vertical gel without SDS. During the IEF, a pH gradient is formed and the charged species move through the gel until they reach a specific pH. In this pH, the proteins have no effective charge (known as protein pI). The IEF shows high resolution, being able to separate macromolecules with pI differences of just 0.001 pH units [54, 55]. In dissociating or denaturing systems, the proteins are solubilized in buffer containing the reagent used to promote protein denaturation. SDS-PAGE, originally described by Laemmli [56], is an electrophoresis technique in polyacrylamide gel (PAGE) that used SDS as a denaturing agent, with interacts with the proteins giving them negative charges, allowing them to migrate, through a polyacrylamide gel towards a positive electrode a be separated by the differences related to their mass

Teixeira et al [25] described the purification of an acid phospholipase from B. pirajai (BpirPLA2I). As a biochemical characterization step, polyacrylamide gel electrophoresis in denaturing conditions (SDS-PAGE) was done. Using this approach, carried out in reducing and non-reducing conditions, the author could infer that the purified protein had the form of a monomer with apparent molecular mass of 14 kDa, both in reducing conditions as well as in non-reducing conditions the proteins presented the same mass, being confirmed afterwards by mass spectrometry.

Moreover, Torres [57] fractionated B. marajoensis venom using a cationic ion exchange column followed by an analytical phase in RP- HPLC, obtaining a phospholipase BmarPLA2 that was submitted to SDS-PAGE in reducing conditions showing apparent molecular mass of 14 kDa. However, in non-reducing conditions, the author observed the appearance of a single band at 28 kDa, concluding that BmarPLA2 was a dimeric structured protein joined by disulphide bridges. Thus, the above-cited examples demonstrate the importance of this procedure (SDS-PAGE) as a protein characterization step.

The determination of the isoelectric point is another important biochemical characterization of phospholipases A2. Previous studies involving phospholipases from snake venoms have shown that the acid phospholipases are catalytically more active than their basic isoforms [22, 42, 58]. Therefore, many authors have included, as a biochemical characterization parameter, the determination of the isoelectric point of the by isoelectric focusing. Due to pI determination importance, Teixeira [25] used the methodology proposed by Vesterberg and Eriksson [59] to evaluate pI of BpirPLA2-1. In order to obtain the pI value, a 7% polyacrylamide gel was prepared and polymerized over a glass plate of 12 x 14 cm using a U shaped rubber as support. A millimeter plate was previously greased with glycerin for better refrigeration of the gel. Two strips of Pharmacia Biotech were used to connect the gel and the platinum electrodes. The cathode was in contact with NaOH 1 M solution and the anode was in phosphoric acid 1 M. The platinum electrodes were centered over the paper strips and the system was then closed. The high voltage source was adjusted to the maximum values of 500 V, 10 mA, 3 watts and 30 minutes for a pre-run. Following, the samples were applied always in the intersection of two blue lines, exactly over the more central line of the gel. Then the source was programed for 1500 V, 15 mA, 10 watts and 5 h. The end of the run was determined when the source showed a high voltage and low amperage (around 1 mA). After isoelectric focusing, about 1 cm width (lengthwise) were sliced from each extremity of the gel and placed in test tubes containing 200 µL of distilled water for the pH reading after 2 hours of rest. Next, the pH gradient determination graph was plotted. The remaining gel containing the samples was fixed in solution of trichloroacetic acid for 30 minutes, followed by silver staining.

Another important technique as a step to characterize components from snake venoms is the bidimensional electrophoresis (2D). This one was initially developed by O'Farrell [60]. The original methodology consisted of the preparation of polyacrylamide cylindrical gels, in which a pH gradient was established through a pre-run with specific amphoterics (also called ampholytes), that present high buffering capability in pHs close to their isoelectric points (pIs). The proteins were then submitted to an isoelectric focusing (IEF) and subsequently to an electrophoresis in the presence of SDS by a conventional system described by Laemmli [56]. Then, proteins were separated in the first dimension according to their pIs (IEF) and in the second dimension based on their molecular mass (SDS-PAGE).

Bidimensional electrophoresis is laborious, time consuming and difficult to be reproduced in different laboratories and depended on the ability of the researcher to obtain consistent results. Nowadays, many of these problems were solved with the development of new technologies. An important advance which has contributed to the increase of the 2D electrophoresis reproducibility was developed by Gorg [61] of the strip form gels with immobilized pH gradient (IPG - immobilized pH gel). The strips are made by the copolymerization of acrylamide with the Immobiline® (Amershan Biosciences/GE Heathcare) reagent, which contains acid and alkaline buffering groups. Another important technological progress was the improvement of the protein samples preparation methods, together with the discovery of new non-ionic detergents, such as CHAPS surfactants and SB 3-10, used with reducing agents adequate for IEF, like Dithiothreitol (DTT) and Tributyl Phosphine (TBP). Studies performed by Herbert [62] demonstrated that these advances had strongly contributed to the solubilization of a greater number of proteins to be analyzed in bidimensional electrophoresis.

The proteomic analysis of snake components has made use of the 2D electrophoresis as a tool, due to its high-resolution capability that allows, in a single process, the determination of apparent molecular mass and isoelectric point of the venom constituents. Fernandez et al. [22] described the determination of the isoelectric point and apparent molecular mass of Basp-PLA2-II using this technique. In order to do it, the protein was focused in IPG Immobiline® Dry Strip of 7 cm and pH 3-10, under a 200 V tension for 1 min, followed by a second stage of 3500 V for 120 minutes. The second dimension was done in SDS-PAGE 12% and then subsequently dyed with Coomassie blue. It was demonstrated that Basp-PLA2 –II had a pI of 4.9, which is close to the theoretical isoelectric point value (pI 5.05) defined by the primary sequence, evaluated using the Compute pI/MW tool ( software and apparent molecular weight between 15 and 16 kDa, consistent with the molecular weight (MW 14,212±6 Da) obtained by ESI/MS (Electrospray Ionization/Mass Spectrometry).

The advantage of this technique is the high resolution. Alape-Giron [63] working with B. asper venom, performed an ontogenic analysis and an analysis based on the snake’s capture location in different regions of Costa Rica. Using tryptic digestion, MALDI-TOF mass fingerprinting analysis and aminoacid sequencing by MALDI-TOF submitted to similarity search by BLAST, the author showed the intra-specific variability in venom composition. It was hence evidenced that among the venoms obtained from adult species collected in the Caribe area and the Pacific area, there are around 30 proteins that are found in a snake group from a place which find no correspondents in the other.

In our lab, this technique has been used as follows: The proteins are separated by the isoelectric point in 13 cm strips with pH values varying between 3 and 10 in a nonlinear form. These strips contain polyacrylamide gel, where the gradient pH is formed by the presence of ampholytes. To re-hydrate the strips, 250 μL of sample [400 μg of proteins plus re-hydration solution (7 M of urea, 2 M of Thiourea, 2% of Triton X-100 (v/v/), 1% of IPG Buffer® (v/v) and DTT)] is applied in a channel of the apparatus over which the strips are set. The strip’s gel is re-hydrated at room temperature for about 12 hours. After this period, the strips are taken to the focusing system in the following conditions: (1) 500 V step until accumulates 500 Vh; (2) 500 to 1000 V gradient until it accumulates 800 Vh; (3) 1000 to 8000 V gradient until it accumulates 11300 Vh and (4) 8000 V step until it accumulates 3000 Vh. In average, the program run during 5.5 hours, but the time of the final step can be lengthened, if the sample does not reach to the end of the strip during the running according the initial program, it could be confirmed by a bromophenol blue line. At the end of focusing, the strips are equilibrated in two steps. On the first, 10 mL of the solution containing 6 M of urea, 2% of SDS (m/v), 30% of glycerol (v/v), 75 mM of Tris-HCl (pH 8,8), 0,002% of bromophenol blue and 1% DTT (m/v) for each strip is used. In the second, the same solution is used, but DTT is replaced by 2.5% of iodoacetamide (m/v). Each strip equilibrium step run during 15 minutes, under light stirring. Following that, the strips are applied on 10 % polyacrylamide gels previously prepared on 180 X 160 X 1.0 mm plates. After each strip and the standard stay appropriately accommodated in the polyacrylamide gel, a 0,5% agarose (m/v) heated (40 °C) solution is added. The agarose polymerization, provides an effective contact between the strip and the gel, thus avoiding the appearance of air bubbles. Protein


Figure 6.

Electrophoretic profile in 2D-PAGE 10%, 13 cm strip pH 3-10 non-linear of proteins from crude venom from Bothrops moojeni. Molecular weight (MW) –Color Plus Prestained Protein Marker – Broad Range (7-175 kDa) (P7709S). Coomassie G-250.

separation, according to molecular mass, is done by applying 25 mA per gel and 100 W during approximately 5.5 hours. After this period, the gel is washed with deionized water. Then, the proteins are fixed using a solution containing acetic acid 10% (v/v) and ethanol 40% (v/v) during one hour. Then, the fixing solution is removed and the gel is washed again with deionized water 3 times during 10 minutes. The proteins present in the gel are exposed using traditional methods for protein coloring, such as Coomassie blue or Silver nitrate. An example of the practical application of this methodology can be seen in Figure 6.

4. Functional characterization

Many biological activities are related to myotoxins with PLA2 structure obtained from snake venoms. In bothropic snake bite accidents and in experimental models with the use of these venoms, the noxious activity induced by these toxins on the striated muscles is striking [64]. The detection of the myotoxic activity associated to the phospholipase activity detection (in the case of Snake venom PLA2 Asp49) is used as an important auxiliary biological marker in the purification procedures, monitoring its presence.

The myotoxic activity assay can be done in two ways: in vivo and in vitro. The analysis can be done through the quantification of the released intracellular enzymes activity to the periphery blood or to the supernatant of the culture medium of cellular lineages. There are two main enzymes used to this end:

Creatine Kinase (EC is a dimeric protein formed by the combination of subunits (B and M) and in its cytosolic form is found in many tissues, especially in skeletal muscle tissue (CK-MM), cardiac (CK-MB) and in the brain (CK-BB).

Lactate dehydrogenase (EC is an enzyme widely distributed in many tissues and organisms. It is presented in the form of homo or hetero tetramers of subunits M and H, being present in muscular tissue in the homotetrameric form of subunit M.

In vivo, the CK activity quantification in murine models has been the most used to assay the presence of myotoxic PLA2, especially due to their low cost, ease of performance and high specificity as skeletal muscular tissue lesion markers when exposed to myotoxins.

As for the In vitro assays, myoblast lineages C2C12 (ATCC CRL-1772), differentiated until the formation of myotubules, have been used as models to assay the cellular toxicity, through the quantification of LDH levels in the supernatant of cell cultures exposed to toxins.

Regarding the phospholipase activity detections, it can be done by direct and indirect methods. Directly, it is possible to detect the presence of PLA2s with the use of chromogenic substrates, such as 4N3OBA(4-nitro-3-octanoyloxybenzoic acid) that induce the formation of detectable product at 425 nm [65] and fluorescent substrates (NBD) coupled to phospholipids that are used to quantitively and qualitatively survey the PLA2s activity isolated from snake venom [23].

Indirectly, the approach used consists in the potentiometric assay of the fatty acids released after the enzymatic hydrolysis of the phospholipids, through the quantification with standard alkaline solution [66]. Moreover, fatty acids released by the enzymatic degradation can be quantified through the alteration of the optical density of the pH indicator solution, such as phenol red [67], brilliant yellow [68] and bromothymol blue [69]. Another indirect method to assay PLA2 activity present in samples consists in the detection of hemolysis induced by lysophospholipids derived from phospholipids submitted to enzymatic digestion. This can be done through the quantification of hemoglobin present in solution or through the visualization of hemolytic halo in agarose matrix with immobilized red blood cells.

4.1. In vivo assay of the myotoxic activity

Mice is used for the in vivo assay of the myotoxic activity. Swiss males weighing between 18 g and 22 g, kept in controlled environment (12 h in the light and 12 h in the dark), with food and water ad libitum up to the moment of use. PBS solubilized sample and control (PBS) are filtered through 44 µm pores immediately prior to use. Reagents for CK activity dosage are prepared and used according to manufacturer’s instructions.

A Sample (50 µL) or control (50 µL) will be injected in mice gastrocnemic muscle using adequate device in order to guarantee a precise volume control. After a time lap (3 and/or 6 h), blood sample is collected in heparinized tubes and centrifuged to separate plasma. CK concentration is determined according to manufacturer’s instructions and expressed in U/L, where one unit corresponds to the production of 1 mmol of NADH per minute [26,70-72].

4.2. In vitro myotoxic activity assay

In order to assay myotoxic in vitro activity, myoblast lineage cells are used, such as murine skeletal muscle C2C12 myoblasts (ATCC CRL-1772) as described by Lomonte et al. [73], cultivated in modified Dubelco Eagle medium, supplemented with 1% bovine fetal serum. PBS solubilized sample, negative control (PBS) and positive control (Triton X-100) should be filtered through 22 µm pore filters immediately prior to use. Reagents for LDH activity dosage are prepared and used according to manufacturer’s instructions.

In 96 well plate, 2X105 cells/150 µL are set, sample and/or control (50 μL) are incubated in humid atmosphere at 37 °C and 5% CO2 for a 3 hour period. Afterwards, collect supernatant aliquot and quantify LDH activity released by cells with cytoplasmic membrane integrity compromised, according to manufacturer’s instructions and expressed in U/I, where one unit corresponds to the production of 1 mmol of lactate per minute.

5. Phospholipasic activity

5.1. 4N3OBA Substrate enzymatic hydrolysis

Phospholipase A2 activity can be measured according to the technique described by Holzer and Mackessy [65], modified for 96 wells plate [74].

Prepare aliquots of 100 µL of 4N3OBA 0.1% solution in acetonitrile and lyophilize. Keep the aliquots at -20° C until ready to use. The color reagent is prepared solubilizing the contents of one aliquot of 4N3OBA in 1ml of reagent containing Tris 10 mM, CaCl2 10 mM, NaCl 100 mM, and pH 8.0. For the test in micro plates, add 180 µL of color reagent and 20 µL of sample or water (blank), incubate the mixture at 37 °C for 5 minutes, measuring the optical density at 425 nm and 600 nm (to correct sample turbidity) at 30 second intervals. The activity will be expressed according to the equation (1) where 1 unit of phospholipase activity corresponds to the production of 1 µmol of 4-nitro-3-hydroxy-benzoic acid per minute.

PLA2 activity [µmol  min1 µg1] = [OD425nmOD 600nm/min] x 0.07862 [umol/OD425nm] / 1/protein (1/µg)

5.2. Enzymatic hydrolysis of fluorescent substrates (NBD)

The phospholipase activity can also be assayed with the used of chromogenic substrates, using acyl-NBD reagents: NBD-PC (Phosphatidylcholine), NBDPG (phosphatidylglycerol), NBD-PE (phosphatidylethanolamine) or NBD-PA (phosphatidic acid). A solution of fluorescent lipids should be previously prepared in a 1 mg/ml concentration in chloroform. 100 µL aliquots are distributed and then dried under nitrogen flow. The dried lipid will be solubilized in 1 ml of NaCl 0.15 M and sonicated until the obtention of a limpid solution. For the test, the lipids should be diluted in a solution containing Tris-HCl 50 mM, CaCl2 1 mM pH 7.5. Initially, incubate the solution at 37 °C and, after 2 minutes, make an initial reading, configuring the equipment for excitation at 460 nm and emission at 534 nm. Following, apply the sample and make a second reading after 12 minutes. The change in fluorescence intensity is converted to nanomoles of product per minute (nmoles/min) using a calibration curve, prepared by hydrolyzing completely a substrate solution through sodium hydroxide treatment. The fluorescence intensity unit was converted to nmoles/min [33].

5.3. Potentiometric titration of fatty acids

The phospholipase activity can be assayed by potentiometric titration as described by de Haas [75], using as substrate an egg yolk emulsion in the presence of sodium deoxycholate 0.03 M and CaCl2 0.6 M. Fatty acids released enzymatically are titrated with a standard solution of NaOH 0.1 N at pH 8.0 at room temperature. The phospholipase activity is generally done with different concentrations of toxin, and calculated per amount of microequivalents of alkali consumed per minute, by mg of protein. One unit of phospholipase activity can be defined as the quantity of enzyme that releases 1 μmol of fatty acid per minute, in the reaction conditions.

5.4. Phenol red

The spectrophotometric detections of phenol red solution, induced by the increase of free fatty acids concentration can also be used to assay the phospholipase activity in samples, as described by Radvanyi [67].

In order to use this technique, prepare the reagent solution containing Phosphatidylcholine 0.25% (w/v) TritonX-100 0.4% (v/v), phenol chloride 32 mM. In a thermostatic cuvette at37 °C, add 1mL of reagent solution and 10 µL of sample. After stabilization for 20 seconds, determine the optical density measuring at 558 nm for 3 minutes, in kinetic intervals of 15 seconds. One unit of phospholipase can be defined as the quantity of enzyme necessary to convert 0.001 UA 558 nm per minute.

5.5. Indirect hemolysis

In this test, phospholipids (from egg yolk, soy lecithin or other sources) are used as substrates, with the production of fatty acid and corresponding lysophospholipids.These lysophospholipids have membrane activity over red blood cells, producing hemolysis that can be detected through the quantification of hemoglobin present in solution or through a hemolysis halo present in agarose gel containing intact red blood cells [76].

For the test, collect blood in a heparinized tube, wash the red blood cells with PBS, centrifuging at 800 xg for 5 minutes and prepare the suspension at 3%. Prepare solution containing phosphate buffer 20 mM, sodium chloride 100 mM and CaCl2 10 mM, erythrocyte suspension 3% (1:30 v/v) and egg yolk solution 0.1% (1:30 v/v). Add 10 µL of sample or PBS (control 0%) or Triton X-100 0.1% (control 100%) and incubate at 37 °C for 30 minutes. Then, centrifuge, collect the supernatant and determine the optical density at 405 nm, using PBS as blank. The results will be expressed in % of hemolysis compared with the positive control.

Hemoglobin dosage present in solution with the use of the Drabkin reagent (potassium ferrocyanide in buffered environment) [77] can be done by comparing the optical density of the samples with the standard curve made with the hemoglobin cyanide solution, according to manufacturer instructions.

To assay the hemolytic activity in agarose gel, carefully heat the suspension containing agarose 2% in PBS until complete fusion. After partial cooling (45 °C), add an equal volume of PBS containing CaCl2 0.02 M; egg yolk suspension (1:30 m/v), erythrocytes washed in PBS (1:30 m/v), pouring over Petri plate until the formation of a layer approximately 2 mm thick. After solidification of the gel, orifices of uniform diameter (0.2 cm diameter) to apply the sample are made. The gel is incubated for 12 hours, at 37 °C and humid environment. The formation of a translucid halo around the gel application point is indicative of phospholipase activity, contrasting with the rest of the gel which remains with a reddish tone due to the presence of integral red blood cells retained in the gel net.


PLA2s: Phospholipases A2sPLA2: secreted PLA2 cPLA2 : cytosolic PLA2iPLA2: Ca2+ independent PLA2 PAF-AH: acetyl-hydrolases from platelet activating factors and liposomalMr: relative massPAF: platelet activating factorssvPLA2s: phospholipase A2 found in snake venomsSDS-PAGE: Sodium dodecyl sulfate – PolyAcrylamide Gel ElectrophoresisMW: Molecular weightRP-HPLC: reverse-phase chromatography DEAE-Sepharose: Dietylaminoetyl Sepharose tEnd cells: endothelial cellsCNBr: cyanogen bromide PBS: phosphate buffer salineIEF: isoelectric focusing2DE: bi-dimensional electrophoresis IPG: immobilized pH gelpIs: isoelectric pointsDTT: Dithiothreitol TBP: Tributyl phosphine ESI: Electrospray IonizationMS: Mass SpectrometryCK-MM: Creatine Kinase - skeletal muscle tissue CK-MB: Creatine Kinase – cardiacCK-BB: Creatine Kinase - brain CK: Creatine KinaseLDH: lactate dehydrogenaseNBD: N-4-Nitrobenzo-2-Oxa-1,3-DiazoleNBD-PC: N-4-Nitrobenzo-2-Oxa-1,3-Diazole PhosphatidylcholineNBD-PG: N-4-Nitrobenzo-2-Oxa-1,3-DiazoleNBD-PE: N-4-Nitrobenzo-2-Oxa-1,3-Diazole phosphatidylglycerolNBD-PA: N-4-Nitrobenzo-2-Oxa-1,3-Diazole Phosphatidic acid


The authors are grateful to Professor Luiz Hildebrando Pereira da Silva, George A. Oliveira, Marjorie J. M. Nascimento and Rafaela D. Souza by the assistance and to the Ministry of Science and Technology (MCT), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/MCT), Financiadora de Estudos e Projetos (FINEP/MCT), Fundação de Tecnologia do Acre/Fundo de Desenvolvimento Científico e Tecnológico (Funtac/FDCT), Secretary of Development of the Rondônia State (PRONEX/CNPq), Instituto Nacional para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica (INCTINPeTAm/CNPq/MCT), and Rede de Biodiversidade e Biotecnologia da Amazônia Legal (Rede Bionorte/MCT) for the financial support.


1 - D. Kordis, F. Gubensek, 2000Adaptive evolution of animal toxin multigene familiesGene14352
2 - A. Menez, 1998Functional architectures of animal toxins: a clue to drug design Toxicon 1115571572
3 - T. S. Kang, D. Georgieva, N. Genov, M. T. Murakami, M. Sinha, R. P. Kumar, P. Kaur, S. Kumar, S. Dey, S. Sharma, A. Vrielink, C. Betzel, S. Takeda, R. K. Arni, T. P. Singh, R. M. Kini, 2011Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis.FEBS J. 2345444576
4 - J. J. Calvete, P. Juarez, L. Sanz, 2007Snake venomics. Strategy and applications. J. Mass Spectrom. 1114051414
5 - D. Mebs, 2001Toxicity in animals. Trends in evolution.Toxicon 18796
6 - E. A. Dennis, J. Cao, Y. H. Hsu, V. Magrioti, G. Kokotos, 2011Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.Chem. Rev. 1061306185
7 - Gutierrez J.M, Ownby C.L, Odell G.V1984Isolation of a Myotoxin from Bothrops asper Venom- Partial Characterization and Action on Skeletal-Muscle. Toxicon 1115128
8 - de Paula R.C, Castro H.C, Rodrigues C.R, Melo P.A, Fuly A.L2009Structural and pharmacological features of phospholipases A2 from snake venoms. Protein Pept. Lett. 8899907
9 - A. M. Soares, W. P. Sestito, S. Marcussi, R. G. Stabeli, S. H. Andriao-Escarso, O. A. Cunha, C. A. Vieira, J. R. Giglio, 2004Alkylation of myotoxic phospholipases A2 in Bothrops moojeni venom: a promising approach to an enhanced antivenom production. Int. J. Biochem. Cell. Biol. 2258270
10 - V. M. Rodrigues, S. Marcussi, R. S. Cambraia, A. L. de Araujo, N. R. Malta-Neto, A. Hamaguchi, E. A. Ferro, M. I. Homsi-Brandeburgo, J. R. Giglio, A. M. Soares, 2004Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom. Toxicon 3305314
11 - S. Taghrid, T. S. Istivan, P. J. Coloe, 2006Phospholipase A in Gram-negative bacteria and its role in pathogenesis. Microbiology 15212631274
12 - M. Waite, 1996Phospholipases. In Biochemistry of Lipids, Lipoproteins and Membranes, 211236Edited by D. E. Vance & J. Vance. Amsterdam: Elsevier.
13 - Dennis E.A1994Diversity of Group Types, Regulation, and Function of Phospholipase A2. J. Biol. Chem. 2691305713060
14 - Dennis E.A1997The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 2212
15 - Arni R.K, Ward R.J1996Phospholipase A2 a structural review. Toxicon 8827841
16 - Burke J.E, Dennis E.A2009Phospholipase A2 biochemistry. Cardiovasc. Drugs Ther. 14959
17 - de Oliveira T.C, de Amorim H.L.N, Guimaraes J.A2007Interfacial activation of snake venom phospholipases A2 svPLA2 probed by molecular dynamics simulations. J. Mol. Struct. Theochem. 1-3: 31-41.
18 - H. M. Verheij, J. J. Volwerk, E. H. J. M. Jansen, W. C. Puyk, B. W. Dijkstra, J. Drenth, G. H. Dehaas, 1980Methylation of Histidine-48 in Pancreatic Phospholipase A2- Role of Histidine and Calcium-Ion in the Catalytic Mechanism. Biochemistry 4743750
19 - dos Santos J.I, Fernandes C.A, Magro A.J, Fontes M.R2009The intriguing phospholipases A2 homologues: relevant structural features on myotoxicity and catalytic inactivity. Protein Pept. Lett. 8887893
20 - B. Lomonte, P. Rey-Suarez, W. C. Tsai, Y. Angulo, M. Sasa, J. M. Gutierrez, J. J. Calvete, 2012Snake venomics of the pit vipers Porthidium nasutum, Porthidium ophryomegas, and Cerrophidion godmani from Costa Rica: toxicological and taxonomical insights. J. Proteomics 516751689
21 - S. H. Andriao-Escarso, A. M. Soares, V. M. Rodrigues, Y. Angulo, C. Diaz, B. Lomonte, J. M. Gutierrez, J. R. Giglio, 2000Myotoxic phospholipases A2 in Bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothrops toxins from Bothrops jararacussu. Biochimie 8755763
22 - J. Fernandez, J. M. Gutierrez, Y. Angulo, L. Sanz, P. Juarez, J. J. Calvete, B. Lomonte, 2010Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: biochemical and toxicological characterization. Biochimie 3273283
23 - R. S. Rodrigues, L. F. Izidoro, S. S. Teixeira, L. B. Silveira, A. Hamaguchi, M. I. Homsi-Brandeburgo, H. S. Selistre-de-Araujo, J. R. Giglio, A. L. Fuly, A. M. Soares, V. M. Rodrigues, 2007Isolation and functional characterization of a new myotoxic acidic phospholipase A2 from Bothrops pauloensis snake venom. Toxicon 1153165
24 - L. B. Silveira, D. P. Marchi-Salvador, N. A. Santos-Filho, F. P. Silva, S. Jr Marcussi, A. L. Fuly, A. Nomizo, Silva. S. L. da, R. G. Stabeli, E. C. Arantes, A. M. Soares, 2012Isolation and expression of a hypotensive and anti-platelet acidic phospholipase A2 from Bothrops moojeni snake venom. J. Pharm. Biomed. Anal. In Press
25 - S. S. Teixeira, L. B. Silveira, Silva. F. M. da, D. P. Marchi-Salvador, F. P. Silva, L. F. Jr Izidoro, A. L. Fuly, M. A. Juliano, Santos. C. R. dos, M. T. Murakami, S. V. Sampaio, Silva. S. L. da, A. M. Soares, 2011Molecular characterization of an acidic phospholipase A2 from Bothrops pirajai snake venom: synthetic C-terminal peptide identifies its antiplatelet region. Arch. Toxicol 1012191233
26 - A. M. Soares, S. H. Andriao-Escarso, Y. Angulo, B. Lomonte, J. M. Gutierrez, S. Marangoni, M. H. Toyama, R. K. Arni, J. R. Giglio, 2000Structural and functional characterization of myotoxin I, a Lys49 phospholipase A(2) homologue from Bothrops moojeni (Caissaca) snake venom. Arch. Biochem. Biophys 1715
27 - K. Hanasaki, H. Arita, 1999Biological and pathological functions of phospholipase A2 receptor. Arch. Biochem. Biophys 2215223
28 - G. Lambeau, M. Lazdunski, 1999Receptors for a growing family of secreted phospholipases A2.Trends Pharmacol. Sci. 4162170
29 - E. Valentin, G. Lambeau, 2000What can venom phospholipases A2 tell us about the functional diversity of mammalian secreted phospholipases A2.Biochimie815 EOF31 EOF
30 - Wixom R.L,2001The beginnings of chromatography- The pioneers (1900-1960), In: Gehrke C.W, Wixom R.L, and Bayer E, Ed(s). Journal of Chromatography Library, Elsevier 64138
31 - P. J. Spencer, S. D. Aird, M. Boni-Mitake, N. Nascimento, J. R. Rogero, 1998A single-step purification of bothropstoxin-1Braz. J. Med. Biol. Res. 911251127
32 - B. Lomonte, J. M. Gutierrez, M. F. Furtado, R. Otero, J. P. Rosso, O. Vargas, E. Carmona, M. E. Rovira, 1990Isolation of basic myotoxins from Bothrops moojeni and Bothrops atrox snake venoms.Toxicon 1011371146
33 - Soares A.M, Rodrigues V.M, Borges M.H, Andriao-Escarso S.H, Cunha O.A, Homsi-Brandeburgo M.I, Giglio J.R1997Inhibition of proteases, myotoxins and phospholipases A2 from Bothrops venoms by the heteromeric protein complex of Didelphis albiventris opossum serum.Biochem. Mol. Biol. Int. 510911099
34 - M. I. Homsi-Brandeburgo, L. S. Queiroz, H. Santo-Neto, L. Rodrigues-Simioni, J. R. Giglio, 1988Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin.Toxicon 7615627
35 - M. F. Pereira, J. C. Novello, A. C. Cintra, J. R. Giglio, E. T. Landucci, B. Oliveira, S. Marangoni, 1998The amino acid sequence of bothropstoxin-II, an Asp-49 myotoxin from Bothrops jararacussu (Jararacucu) venom with low phospholipase A2 activity.J. Protein Chem. 4381386
36 - V. M. Rodrigues, S. Marcussi, R. S. Cambraia, A. L. de Araujo, N. R. Malta-Neto, A. Hamaguchi, E. A. Ferro, M. I. Homsi-Brandeburgo, J. R. Giglio, A. M. Soares, 2004Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom. Toxicon 3305314
37 - Rodrigues V.M, Soares A.M, Mancin A.C, Fontes M.R, Homsi-Brandeburgo M.I, Giglio J.R1998Geographic variations in the composition of myotoxins from Bothrops neuwiedi snake venoms: biochemical characterization and biological activityComp. Biochem. Physiol. A Mol. Integr. Physiol. 3215222
38 - D. A. Higuchi, C. M. Barbosa, C. Bincoletto, J. R. Chagas, A. Magalhaes, M. Richardson, E. F. Sanchez, J. B. Pesquero, R. C. Araujo, J. L. Pesquero, 2007Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom.Biochimie3319328
39 - J. J. Daniele, I. D. Bianco, C. Delgado, D. B. Carrillo, G. D. Fidelio, 1997A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarara chica) venom with novel kinetic and chromatographic properties.Toxicon 812051215
40 - Janson J.C1967Adsorption phenomena on Sephadex.J. Chromatogr. 11220
41 - Williams K.W1972Solute-gel interactions in gel filtration.Lab. Pract. 9667670
42 - N. A. Santos-Filho, L. B. Silveira, C. Z. Oliveira, C. P. Bernardes, D. L. Menaldo, A. L. Fuly, E. C. Arantes, S. V. Sampaio, C. C. Mamede, M. E. Beletti, F. de Oliveira, A. M. Soares, 2008A new acidic myotoxic, anti-platelet and prostaglandin I2 inductor phospholipase A2 isolated from Bothrops moojeni snake venomToxicon8908917
43 - D. C. Nunes, R. S. Rodrigues, M. N. Lucena, C. T. Cologna, A. C. Oliveira, A. Hamaguchi, M. I. Homsi-Brandeburgo, E. C. Arantes, D. N. Teixeira, C. Ueira-Vieira, V. M. Rodrigues, 2011Isolation and functional characterization of proinflammatory acidic phospholipase A2 from Bothrops leucurus snake venom. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 3226233
44 - P. A. Melo, G. Suarez-Kurtz, 1988Release of sarcoplasmic enzymes from skeletal muscle by Bothrops jararacussu venom: antagonism by heparin and by the serum of South American marsupials.Toxicon 18795
45 - P. A. Melo, M. I. Homsi-Brandeburgo, J. R. Giglio, G. Suarez-Kurtz, 1993Antagonism of the myotoxic effects of Bothrops jararacussu venom and bothropstoxin by polyanions.Toxicon 3285291
46 - B. Lomonte, E. Moreno, A. Tarkowski, L. A. Hanson, M. Maccarana, 1994Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling.J. Biol. Chem. 472986729873
47 - Stabeli R.G, Magalhaes L.M, Selistre-de-Araujo H.S, Oliveira E.B2005Antibodies to a fragment of the Bothrops moojeniamino acid oxidase cross-react with snake venom components unrelated to the parent protein. Toxicon 3308317
48 - D. G. Beghini, Cruz. da-Hofling, M. A. Randazzo-Moura, P. Rodrigues-Simioni, L. Novello, J. C. Hyslop, S. Marangoni, S. , 2005Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against crotoxin and phospholipase A2 from Crotalus durissus cascavella venomToxicon6604611
49 - P. C. Gomes, Avila. R. A. Machado de, Maria. W. Selena, M. Richardson, C. L. Fortes-Dias, C. Chavez-Olortegui, 2007The co-purification of a lectin (BJcuL) with phospholipases A2 from Bothrops jararacussu snake venom by immunoaffinity chromatography with antibodies to crotoxinToxicon810991108
50 - C. O. Rock, F. Snyder, 1975Metabolic interrelations of hydroxy-substituted ether-linked glycerolipids in the pink portion of the rabbit harderian gland.Arch. Biochem. Biophys 2631636
51 - R. Dijkman, S. H. Beiboer, H. M. Verheij, 1997An affinity column for phospholipase A2 based on immobilised acylaminophospholipid analogues.Biochim. Biophys Acta 118
52 - J. J. Calvete, L. Sanz, Y. Angulo, B. Lomonte, J. M. Gutierrez, 2009bVenoms, venomics, antivenomics. FEBS Lett. 1117361743
53 - J. L. T. Gomez-Ariza, Garcia-Barrera, 2005Analytical Characterization of bioactive metal species in the cellular domain (Metallomics) to simplify environmental and biological proteomicsInt. J. Environ. Anal. Chem. 85255266
54 - Y. X. Gao, C. Y. Chen, P. Q. Zhang, Z. F. Chai, W. He, Y. Y. Huang, 2003Detection of metalloproteins in human liver cytosol by synchrotron radiation X-ray fluorescence after sodium dodecyl sulphate polyacrylamide gel electrophoresisAnal. Chim. Acta 1131137
55 - J. Szpunar, 2004Metallomics: a new frontier in analytical chemistry. Anal. Bioanal Chem. 15456
56 - Laemmli U.K1970Cleavage of Structural Proteins during Assembly of Head of Bacteriophage-T4.Nature 5259: 680.
57 - A. F. C. Torres, R. T. Dantas, M. H. Toyama, E. Diz, F. J. Zara, M. G. R. de Queiroz, N. A. P. Nogueira, M. R. de Oliveira, D. D. Toyama, H. S. A. Monteiro, A. M. C. Martins, 2010Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and L-amino acid oxidase. Toxicon 4795804
58 - Andriao-Escarso S.H, Soares A.M, Fontes M.R, Fuly A.L, Correa F.M, Rosa J.C, Greene L.J, Giglio J.R2002Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A2 from Bothrops jararacussu snake venom. Biochem. Pharmacol. 4723732
59 - O. Vesterberg, R. Eriksson, 1972Detection of fibrinolytic activity by a zymogram technique after isoelectric focusing.Biochim. Biophys. Acta 2393397
60 - O’Farrell P.H1975High-resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1040074021
61 - A. Gorg, W. Postel, J. Weser, W. Patutschnick, H. Cleve, 1985Improved resolution of PI (alpha 1-antitrypsin) phenotypes by a large-scale immobilized pH gradient.Am. J. Hum. Genet. 5922930
62 - B. Herbert, 1999Advances in protein solubilization for two-dimensional electrophoresis. Electrophoresis 4-5: 660-663
63 - A. Alape-Giron, M. Flores-Diaz, L. Sanz, M. Madrigal, J. Escolano, M. Sasa, J. J. Calvete, 2009Studies on the venom proteome of Bothrops asper: perspectives and applicationsToxicon7938948
64 - J. M. Gutierrez, B. Lomonte, 1995Phospholipase A2 myotoxins from Bothrops snake venoms.Toxicon 1114051424
65 - M. Holzer, S. P. Mackessy, 1996An aqueous endpoint assay of snake venom phospholipase A2.Toxicon 1011491155
66 - N. Martin-Mounot, H. Rochat, 1979Isolation and characterization of a toxic phospholipase A2 in the spitting cobra (Naja mossambicamossambica) venom. Toxicon 1712736
67 - F. Radvanyi, B. Saliou, C. Bon, P. N. Strong, 1987The interaction between the presynaptic phospholipase neurotoxins beta-bungarotoxin and crotoxin and mixed detergent-phosphatidylcholine micelles. A comparison with non-neurotoxic snake venom phospholipases A2.J. Biol. Chem. 1989668974
68 - Y. Yao, M. H. Wang, K. Y. Zhao, C. C. Wang, 1998Assay for enzyme activity by following the absorbance change of pH-indicatorsJ. Biochem. Biophys. Methods 2-3: 119 EOF
69 - Price J.A2007A colorimetric assay for measuring phospholipase A2 degradation of phosphatidylcholine at physiological pH.J. Biochem. Biophys Methods 3441444
70 - A. M. Soares, R. Guerra-Sá, C. Borja-Oliveira, V. Rodrigues, L. Rodrigues-Simioni, V. Rodrigues, M. R. M. Fontes, J. R. Giglio, 2000Molecular cloning and functional characterization of BnSP-7, a myotoxin Lys-49 phospholipase A2 homologue, from Bothrops neuwiedi pauloensis venom. Arch. Biochem. Biophys. 378201209
71 - Soares A.M, Mancin A.C, Cecchini A.L, Arantes E.C, Franca S.C, Gutiérrez J.M, Giglio J.R2001Effects of chemical modifications of crotoxin B, the phospholipase A2 subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activitiesInt. J. Biochem. Cell Biol. 33877888
72 - A. M. Soares, S. H. Andriao-Escarso, R. K. Bortoleto, L. Rodrigues-Simioni, R. K. Arni, R. J. Ward, J. M. Gutiérrez, J. R. Giglio, 2001Dissociation of enzymatic and pharmacological properties of piratoxins-I and-III, two myotoxic phospholipases A2 from Bothrops pirajai snake venom. Arch. Biochem. Biophys. 38718819
73 - B. Lomonte, Y. Angulo, S. Rufini, W. Cho, J. R. Giglio, M. Ohno, J. J. Daniele, P. Geoghegan, J. M. Gutierrez, 1999Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro.Toxicon 37145158
74 - L. A. Ponce-Soto, V. L. Bonfim, L. Rodrigues-Simioni, J. C. Novello, S. Marangoni, 2006Determination of primary structure of two isoforms 6-1 and 6-2 PLA2 D49 from Bothrops jararacussu snake venom and neurotoxic characterization using in vitro neuromuscular preparationProtein J. 254755
75 - G. H. de Haas, M. M. Postema, W. Niewenhuizen, L. L. Van Deenen, 1968Purification and properties of phospholipase A and its zymogen from porcine pancreas.Bull. Soc. Chim. Biol. (Paris) 9: 1383 EOF
76 - J. M. Gutierrez, C. Avila, E. Rojas, L. Cerdas, 1988An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica.Toxicon 4411413
77 - Assendelft O.W.Van1970Spectrophotometry of haemoglobin derivativesRoyal Vangorcum Ltd. Assen. The Netherlands and Charles C. Thomas Springfield. III: 152.
78 - T. Nikai, Y. Komori, S. Yagihashi, A. Ohara, Y. Ohizumi, H. Sugihara, 1994Isolation and Characterization of Phospholipase-A2 from Agkistrodon bilineatus (Common Cantil) Venom.Int. J. Biochem. 26879884
79 - J. Takagi, F. Sekiya, K. Kasahara, Y. Inada, Y. Saito, 1988Venom from southern copperhead snake Agkistrodon contortrix contortrix II. A unique phospholipase A2 that induces platelet aggregation. Toxicon 2199206
80 - Johnson E.K, Ownby C.L1993Isolation of a myotoxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copperhead) and pathogenesis of myonecrosis induced by it in mice.Toxicon 3243255
81 - R. S. Leite, W. Franco, C. L. Ownby, H. S. Selistre-de-Araujo, 2004Effects of ACL myotoxin, a Lys49 phospholipase A2 from Agkistrodon contortrix laticinctus snake venom, on water transport in the isolated toad urinary bladder. Toxicon 17783
82 - E. Rojas, P. Saravia, Y. Angulom, V. Arce, B. Lomonte, J. J. Chavez, R. Velasquez, M. Thelestam, J. M. Gutierrez, 2001Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization, isolation of a myotoxic phospholipase A2 homologue and neutralization by two antivenoms. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 2151162
83 - J. M. Gutierrez, B. Lomonte, L. Cerdas, 1986Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer.Toxicon 9885894
84 - Y. Angulo, T. Olamendi-Portugal, A. Alape-Giron, L. D. Possani, B. Lomonte, 2002Structural characterization and phylogenetic relationships of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a Lys49 phospholipase A2 homologue. Int. J. Biochem. Cell. Biol. 1012681278
85 - Y. Angulo, E. Chaves, A. Alape, A. Rucavado, J. M. Gutierrez, B. Lomonte, 1997Isolation and characterization of a myotoxic phospholipase A2 from the venom of the arboreal snake Bothriechis (Bothrops) schlegelii from Costa Rica.Arch. Biochem. Biophys 2260266
86 - S. Huancahuire-Veja, L. A. Ponce-Soto, D. Martins-de-Souza, S. Marangoni, 2011Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 2108119
87 - H. S. Selistre, L. S. Queiroz, O. A. Cunha, G. E. De Souza, J. R. Giglio, 1990Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom.Toxicon 3261273
88 - J. C. Cogo, S. Lilla, G. H. Souza, S. Hyslop, G. de Nucci, 2006Purification, sequencing and structural analysis of two acidic phospholipases A2 from the venom of Bothrops insularis (jararaca ilhoa).Biochimie1219471959
89 - Machado Braga M.D, Costa Martins A.M, Alves C.D, de Menezes D.B, Martins R.D, Ferreira Barbosa P.S, de Sousa Oliveira I.M, Toyama M.H, Toyama D.O, Dos Santos Diz Filho E.B, Ramos Fagundes F.H, Fonteles M.C, Azul Monteiro H.S2008Purification and renal effects of phospholipase A2 isolated from Bothrops insularis venom. Toxicon 2181190
90 - S. M. Serrano, A. P. Reichl, R. Mentele, E. A. Auerswald, M. L. Santoro, C. A. Sampaio, A. C. Camargo, M. T. Assakura, 1999A novel phospholipase A2, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor.Arch. Biochem. Biophys. 12632
91 - Machado O.L, Oliveira-Carvalho A.L, Zingali R.B, Carlini C.R1993Purification physicochemical characterization and N-terminal-amino acid sequence of a phospholipase A2 from Bothrops jararaca venom. Braz. J. Med. Biol. Res. 2163166
92 - Denegri. M. E. Garcia, O. C. Acosta, S. Huancahuire-Veja, D. Martins-de-Souza, S. Marangoni, S. L. Marunak, G. P. Teibler, L. C. Leiva, L. A. Ponce-Soto, 2010Isolation and functional characterization of a new acidic PLA2 Ba SpII RP4 of the Bothrops alternatus snake venom from ArgentinaToxicon16474
93 - H. E. Nisenbom, C. Seki, J. C. Vidal, 1986Phospholipase A2 from Bothrops alternatus (vibora de la cruz) venom. Purification and some characteristic properties.Toxicon 3259272
94 - L. A. Ponce-Soto, B. Lomonte, J. M. Gutierrez, L. Rodrigues-Simioni, J. C. Novello, S. Marangoni, 2007aStructural and functional properties of BaTX, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus.Biochim. Biophys. Acta 4585593
95 - A. Quintero, A. M. Soares, 2010Functional and structural characterization of phospholipases A2 isolated from Bothrops asper snake venom in Panama.J. Venom. Anim. Toxins incl. Trop. Dis. 4664664
96 - B. Lomonte, J. M. Gutierrez, 1989A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo).Toxicon 27725733
97 - I. I. Kaiser, J. M. Gutierrez, D. Plummer, S. D. Aird, G. V. Odell, 1990The Amino-Acid-Sequence of a Myotoxic Phospholipase from the Venom of Bothrops asper. Arch. Biochem. Biophys 2319325
98 - D. Mebs, Y. Samejima, 1986Isolation and characterization of myotoxic phospholipases A2 from crotalid venoms.Toxicon 2161168
99 - M. M. Kanashiro, M. E. R. de Cassia, J. H. Petretski, M. V. Prates, E. W. Alves, O. L. Machado, Silva. W. D. da, T. L. Kipnis, 2002Biochemical and biological properties of phospholipases A2 from Bothrops atrox snake venom. Biochem. Pharmacol. 711791186
100 - V. Nunez, V. Arce, J. M. Gutierrez, B. Lomonte, 2004Structural and functional characterization of myotoxin I, a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops atrox.Toxicon 191101
101 - T. R. Costa, D. L. Menaldo, C. Z. Oliveira, N. A. Santos-Filho, S. S. Teixeira, A. Nomizo, A. L. Fuly, M. C. Monteiro, B. M. de Souza, M. S. Palma, R. G. Stabeli, S. V. Sampaio, A. M. Soares, 2008Myotoxic phospholipases A2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: cytotoxic effect on microorganism and tumor cells. Peptides 1016451656
102 - S. Huancahuire-Vega, L. A. Ponce-Soto, D. Martins-de-Souza, S. Marangoni, 2009Structural and functional characterization of brazilitoxins II and III (BbTX-II and-III), two myotoxins from the venom of Bothrops brazili snake. Toxicon 6818827
103 - Modesto. J. C. de Albuquerque, P. J. Spencer, M. Fritzen, R. C. Valenca, M. L. Oliva, Silva. M. B. da, A. M. Chudzinski-Tavassi, M. C. Guarnieri, 2006BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom: isolation, cloning and characterization as potent anti-platelet and inductor of prostaglandin I2 release by endothelial cells.Biochem. Pharmacol. 3377384
104 - V. L. Bonfim, M. H. Toyama, J. C. Novello, S. Hyslop, C. R. Oliveira, L. Rodrigues-Simioni, S. Marangoni, 2001Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venomJ Protein Chem 3239245
105 - D. F. Ketelhut, M. H. de Mello, E. L. Veronese, L. E. Esmeraldino, M. T. Murakami, R. K. Arni, J. R. Giglio, A. C. Cintra, S. V. Sampaio, 2003Isolation, characterization and biological activity of acidic phospholipase A2 isoforms from Bothrops jararacussu snake venom.Biochimie10983991
106 - J. M. Gutierrez, L. Sanz, J. Escolano, J. Fernandez, B. Lomonte, Y. Angulo, A. Rucavado, D. A. Warrell, J. J. Calvete, 2008Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenomJ. Proteome Res. 1043964408
107 - L. A. Ponce-Soto, D. Martins-de-Souza, S. Marangoni, 2010Neurotoxic, myotoxic and cytolytic activities of the new basic PLA2 isoforms BmjeTX-I and BmjeTX-II isolated from the Bothrops marajoensis (Marajo Lancehead) snake venom. Protein J. 2103113
108 - Torres. A. F. Costa, R. T. Dantas, M. H. Toyama, Filho. E. Diz, F. J. Zara, Queiroz. M. G. Rodrigues de, Nogueira. N. A. Pinto, Oliveira. M. Rosa de, Toyama. D. de Oliveira, H. Monteiro, S, Martins A.M (2010Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and L-amino acid oxidase. Toxicon 4795804
109 - C. Galbiatti, T. Rocha, P. Randazzo-Moura, L. A. Ponce-Soto, S. Marangoni, M. A. Cruz-Höfling, L. Rodrigues-Simioni, 2012Pharmacological and partial biochemical characterization of Bmaj-9 isolated from Bothrops marajoensis snake venomJ. Venom. Anim. Toxins incl. Trop. Dis 186272
110 - A. M. Perchuc, L. Menin, P. Favreau, B. Buhler, P. Bulet, R. Schoni, M. Wilmer, B. Ernst, R. Stocklin, 2010Isolation and characterization of two new Lys49 PLA2s with heparin neutralizing properties from Bothrops moojeni snake venomToxicon610801092
111 - Soares A.M, Rodrigues V.M, Homsi-Brandeburgo M.I, Toyama M.H, Lombardi F.R, Arni R.K, Giglio J.R1998A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: biochemical characterization, crystallization, myotoxic and edematogenic activity.Toxicon 3503514
112 - A. K. Calgarotto, D. C. Damico, L. A. Ponce-Soto, P. A. Baldasso, Silva. S. L. Da, G. H. Souza, M. N. Eberlin, S. Marangoni, 2008Biological and biochemical characterization of new basic phospholipase A(2) BmTX-I isolated from Bothrops moojeni snake venom. Toxicon 815091519
113 - M. R. Queiroz, C. C. Mamede, K. C. Fonseca, L. C. M. N. Canabrava, L. V. Franca, M. C. Silva, L. Stanziola, M. E. Beletti, H. A. N. Canabrava, F. Oliveira, 2011Biological characterization of a myotoxin phosphoplipase A2 homologue purified from the venom of the snake Bothrops moojeni. J. Venom.Anim. Toxins incl. Trop. Dis.14958
114 - L. C. Mancuso, M. M. Correa, C. A. Vieira, O. A. Cunha, J. Lachat, H. S. de Araujo, C. L. Ownby, J. R. Giglio, 1995Fractionation of Bothrops pirajai snake venom: isolation and characterization of piratoxin-I, a new myotoxic proteinToxicon5615626
115 - M. H. Toyama, P. D. Costa, J. C. Novello, B. de Oliveira, J. R. Giglio, Cruz. da-Hofling, M. A. Marangon, S. , 1999Purification and amino acid sequence of MP-III 4R D49 phospholipase A2 from Bothrops pirajai snake venom, a toxin with moderate PLA2 and anticoagulant activities and high myotoxic activity. J. Protein Chem. 3371378
116 - M. H. Toyama, L. C. Mancuso, J. R. Giglio, J. C. Novello, B. Oliveira, S. Marangoni, 1995A quick procedure for the isolation of dimeric piratoxins-I and II, two myotoxins from Bothrops pirajai snake venom.N-terminal sequencing.Biochem. Mol. Biol. Int. 610471055
117 - C. Diaz, J. M. Gutierrez, B. Lomonte, 1992Isolation and Characterization of Basic Myotoxic Phospholipases-A2 from Bothrops Godmani (Godman Pit Viper) Snake-Venom. Arch. Biochem. Biophys 1135142
118 - M. V. de Sousa, L. Morhy, R. K. Arni, R. J. Ward, C. Diaz, J. M. Gutierrez, 1998Amino acid sequence of a myotoxic Lys49-phospholipase A2 homologue from the venom of Cerrophidion (Bothrops) godmaniBiochim. Biophys Acta 2204208
119 - Tsai I.H, Chen Y.H, Wang Y.M, Tu M.C, Tu A.T2001Purification, sequencing, and phylogenetic analyses of novel Lys-49 phospholipases A2 from the venoms of rattlesnakes and other pit vipers. Arch. Biochem. Biophys 2236244
120 - F. V. Fonseca, E. Antunes, R. P. Morganti, H. S. Monteiro, A. M. Martins, D. O. Toyama, S. Marangoni, M. H. Toyama, 2006Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venomProtein J. 3183192
121 - L. A. Ponce-Soto, P. A. Baldasso, F. F. Romero-Vargas, F. V. Winck, J. C. Novello, S. Marangoni, 2007Biochemical pharmacological and structural characterization of two PLA2 isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom. Protein J. 13949
122 - F. F. Romero-Vargas, L. A. Ponce-Soto, D. Martins-de-Souza, S. Marangoni, 2010Biological and biochemical characterization of two new PLA2 isoforms Cdc-9 and Cdc-10 from Crotalus durissus cumanensis snake venom. Comp. Biochem. Physiol. C. Toxicol Pharmacol. 16674
123 - J. A. Pereanez, V. Nunez, S. Huancahuire-Vega, S. Marangoni, L. A. Ponce-Soto, 2009Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis. Toxicon 5534542
124 - Filho. E. B. Diz, S. Marangoni, D. O. Toyama, F. H. Fagundes, S. C. Oliveira, F. V. Fonseca, A. K. Calgarotto, P. P. Joazeiro, M. H. Toyama, 2009Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruimaToxicon1104114
125 - S. Hernandez-Oliveira, M. H. Toyama, D. O. Toyama, S. Marangoni, S. Hyslop, L. Rodrigues-Simioni, 2005Biochemical, pharmacological and structural characterization of a new PLA2 from Crotalus durissus terrificus (South American rattlesnake) venom. Protein J. 4233242
126 - G. Faure, C. Bon, 1987Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus.Toxicon 2229234
127 - D. G. Oliveira, M. H. Toyama, J. C. Novello, L. O. Beriam, S. Marangoni, 2002Structural and functional characterization of basic PLA2 isolated from Crotalus durissus terrificus venom.J. Protein Chem. 3161168
128 - M. H. Toyama, D. G. de Oliveira, L. O. Beriam, J. C. Novello, L. Rodrigues-Simioni, S. Marangoni, (2003). Structural, enzymatic and biological properties of new PLA2 isoform from Crotalus durissus terrificus venom. Toxicon 810331038 .
129 - H. Zepeda, E. D. Rael, R. A. Knight, 1985Isolation of two phospholipases A2 from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom and variation of immunologically related venom proteins in different populations.Comp. Biochem. Physiol. B. 2319324
130 - D. C. Damico, L. G. Bueno, L. Rodrigues-Simioni, S. Marangoni, Cruz. da-Hofling, M. A. Novello, J. C. , 2006Functional characterization of a basic D49 phospholipase A2 (LmTX-I) from the venom of the snake Lachesis muta muta (bushmaster)Toxicon7759765
131 - Fuly A.L, de Miranda A.L, Zingali R.B, Guimaraes J.A2002Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom.Biochem. Pharmacol. 915891597
132 - E. B. de Assis, M. I. Estevao-Costa, do Valentim. A. Carmo, A. Silva-Neto, Cotta. G. Agostini, Mudado. M. Alvarenga, M. Richardson, C. L. Fortes-Dias, 2008Purification and complete primary structure of the first PLA2 from Lachesis stenophrys (the Central American Bushmaster) snake venom. Protein J5327333
133 - L. J. Vargas, M. Londono, J. C. Quintana, C. Rua, C. Segura, B. Lomonte, V. Nunez, 2012An acidic phospholipase A2 with antibacterial activity from Porthidium nasutum snake venom. Comp Biochem. Physiol. B. Biochem. Mol. Biol. 4341347
134 - C. J. Bohlen, A. T. Chesler, R. Sharif-Naeini, K. F. Medzihradszky, S. Zhou, Sanchez. E. E. King, A. L. Burlingame, A. I. Basbaum, D. Julius, 2011A heteromeric Texas coral snake toxin targets acid-sensing ion channels to produce pain.Nature7373410414
135 - Possani L.D, Alagon A.C, Fletcher P.L, Jr, Varela M.J, Julia J.Z1979Purification and characterization of a phospholipase A2 from the venom of the coral snake, Micrurus fulvius microgalbineus (Brown and Smith).Biochem J. 3603606
136 - J. C. Dokmetjian, S. Del Canto, S. Vinzon, Bonino. M. B. de Jimenez, 2009Biochemical characterization of the Micrurus pyrrhocryptus venom. Toxicon 3375382
137 - A. Alape-Giron, B. Persson, E. Cederlund, M. Flores-Diaz, J. M. Gutierrez, M. Thelestam, T. Bergman, H. Jornvall, 1999Elapid venom toxins: multiple recruitments of ancient scaffolds.Eur. J. Biochem. 1-2: 225 EOF34 EOF
138 - J. Mochca-Morales, B. M. Martin, L. D. Possani, 1990Isolation and characterization of helothermine, a novel toxin from Heloderma horridum horridum (Mexican beaded lizard) venom.Toxicon 3299309
139 - Belo. C. A. Dal, M. H. Toyama, D. Toyama, S. Marangoni, F. B. Moreno, B. S. Cavada, M. D. Fontana, S. Hyslop, E. M. Carneiro, A. C. Boschero, 2005Determination of the amino acid sequence of a new phospholipase A(2) (MIDCA1) isolated from Micrurus dumerilii carinicauda venom. Protein J. 3147153