PLA2s isolated from American snake venoms and respective chromatographic methods used.
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 .
One of the most important protein super-families present in snake venoms are the phospholipases A2 (PLA2, E.C. 22.214.171.124), 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 . According to Waite , 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 .
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 , 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
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
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 .
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
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  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.  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
Some authors have proposed changes to the methodology described above. Spencer et al.  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
Resolution differences were also observed by other authors. As performed by Lomonte et al. , the isolation of two basic myotoxins, MjTX-I e MjTX-II, from the
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.  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.  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. .
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.  described the isolation of two PLA2s isoforms from the
Also, with the use of a multiple step procedure  successfully isolated two isoforms of PLA2s from
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.  described the fractioning of the
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. , working with
In a work published in 2011, Nunes et al.  described the isolation of an acid phospholipase named BL-PLA2, obtained from
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
Following this strategy, Soares et al.  described the purification of BnSP-7, a myotoxin Lys-49 from
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.  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
Based on this information, pertinent to the crossed immunoreactivity existent between venom components, Gomes et al.  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
Different authors used substrate analogous or reversible inhibitors coupled to the chromatographic resin. Rock and Snyder  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  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
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  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 . 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 , 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  described the purification of an acid phospholipase from
Moreover, Torres  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  used the methodology proposed by Vesterberg and Eriksson  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 . 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 . 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  of the strip form gels with immobilized pH gradient (IPG -
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.  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  working with
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
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 . 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:
Creatine Kinase (EC 126.96.36.199): 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 188.8.131.52): 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.
As for the
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  and fluorescent substrates (NBD) coupled to phospholipids that are used to quantitively and qualitatively survey the PLA2s activity isolated from snake venom .
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 . 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 , brilliant yellow  and bromothymol blue . 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
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].
In vitro myotoxic activity assay
In order to assay myotoxic
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 , modified for 96 wells plate .
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.
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 .
5.3. Potentiometric titration of fatty acids
The phospholipase activity can be assayed by potentiometric titration as described by de Haas , 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 .
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 .
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)  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.Abbreviations
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.
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