Purification of Phospholipases A 2 from American Snake Venoms

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].


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 compose d by proteins, comprising a variety of enzymes, such as proteases (metalo and serine), phospholipases A2, L-aminoacid oxidases, esterases, and others [1][2][3][4][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. 3.1.1.4), 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][2][3][4][5][6][7][8][9][10]. 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][13][14]. 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 Ca 2+ 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].
Purification of Phospholipases A2 from American Snake Venoms 5 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][22][23][24][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][28][29].

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.  Bothrops pirajai Piratoxin -II Absence 13.7 9.0 P82287 Gel filtration chromatography on Sephadex G-75® column and ion-exchange chromatography on Sephadex C25® column. [116] Purification of Phospholipases A2 from American Snake Venoms 9 Ion-exchange chromatography on CM-Sephadex® column. [117] Cerrophidion goodmani GodMT-II Absence 13.7 Ion-exchange chromatography on CM-Sephadex® column.
[121] Ion-exchange chromatography on Mono Q FF® column followed by reverse phase chromatography on Vydac® C4 column.

MiDCA1
Presence 15.5 8.0 Reverse phase chromatography on Sephasil Peptide® C18 column followed by reverse phase chromatography on µ-Bondapak® C18 column. [139] 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).  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. 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 rechromatographed 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 rechromatographed 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][45][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.

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 . 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 Purification of Phospholipases A2 from American Snake Venoms 17 [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 . I n o r d e r t o o b t a i n t h e p I v a l u e , 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 (IPGimmobilized 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 (www.expasy.ch/tools) 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 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.

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 2.7.3.2): 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 1.1.1.27): 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.

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][71][72].

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, 2X10 5 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.

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.

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].

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.

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 at 37 °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.

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