**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. 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 [1-10].

© 2012 Calderon et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Calderon et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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].

Purification of Phospholipases A2 from American Snake Venoms 3

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

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

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

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 βcarboxylic 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

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

venoms of the Viperidae family (subfamily Viperinae and Crotalinae) [15,16].

origin, aminoacid sequence and catalytic mechanisms, among others.

sign and N-bond glycosylation sites [6].

sphere of Ca2+ forming a pentagonal pyramid [9,15].

**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.

2 Chromatography – The Most Versatile Method of Chemical Analysis

and PS in the inner leaflet [11].

general lipid metabolism [12-14].

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

**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 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 βcarboxylic 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].

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

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

Evidences suggest that these activities can be mediated by interactions between PLA2s and

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

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

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

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

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

enzymes [21-25].

mechanisms of action.

**2. PLA2 purification** 

endogen acceptors on the target cell membrane [27-29].

well as possible applications in biotechnological models.

functionality or physical-chemical stability.

could make the purification processes easier.

composition that are ionized as a function of pH.

anion exchange resin.

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].
