**2. PLA2 purification**

4 Chromatography – The Most Versatile Method of Chemical Analysis

released together with the fatty acid [15,17].

reactive intermediary [15].

group of the substrate [18].

activities [9,20].

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

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

**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

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

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

to the structural conformation stability of these enzymes [9,15,19].

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

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

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

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

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

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

Purification of Phospholipases A2 from American Snake Venoms 7

then submitted to íon-exchange on CM-Cellulose®

column, followed by affinity chromatography with immobilized BSA and then submitted to gel filtration on

5PW® column and then submitted to cation-exchange on

and then submitted to ion-exchange on CM-Sepharose

column and then submitted to SP-Sephadex C25®

and then submitted to cation-exchange on Protein pack SP-5PW® column and Reverse Phase chromatography on

submitted to reverse phase on Pep-RPC HR 5/5® column.

column followed by Phenyl-Sepharose CL-4B® column and then submitted to reverse phase chromatography on

followed by reverse phase chromatography on C18 column.

column followed by ion-exchange on SP Sephadex C-50®

column and then submitted to Reverse Phase chromatography on Ultrapore RPRC-C3® column.

column or heparin agarose® column.

column and then submitted to gel filtration chromatography on Sephadex G-75® column.

 Ion-exchange chromatography on CM-Sepharose® column followed by hydrophobic interaction chromatography on Phenyl-Sepharose® column.

Bondapack® C18 column.

Sephadex G-75® column.

cellulose® column.

column.

column.

Presence 14.1 nd Gel filtration chromatography on Sephadex G-75®

followed by reverse phase chromatography on µ-

column followed by gel filtration chromatography on

followed by ion-exchange chromatography on CM-

chromatography on DEAE Sepharose® column, active fractions subjected to reverse phase chromatography on C8 column and finally chromatography with CM-

Reverse Phase chromatography on Vydac® C18 column. [88]

Cellulofine GCL-2000® column.

Protein Pak SP-SPW® column.

Purification strategy Ref.

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[89]

[90]

[91]

[23]

[26]

[92]

[93]

[94]

[95]

[7]

[96]

[97]

[98]

[22]

Species PLA2

*Agkistrodon contortrix contortrix* 

*Agkistrodon contortrix laticinctus* 

*Agkistrodon contortrix laticinctus* 

*Bothriechis (Bothrops) schlegelii* 

*Bothropoides insularis* BinTX-I

*Bothrops asper* MTX-I

*Bothrops asper* Myotoxic

MTX-II MTX-III MTX-IV Basp-I-PLA2

PLA2

BinTX-II

Presence Presence

Presence Absence Presence Absence Presence 14.1 14.2 14.2 Nd 14.2

13.9 13.7

PLA2 Activity

MW (kDa) pI

*Agkistrodon bilineatus* PLA2 Absence 14.0 10.2 Q9PSF9 Gel filtration chromatography on Sephadex G-75® and

*Atropoides nummifer* Myotoxin IH Absence 16.0 Cation-exchange chromatography on CM-Sephadex C-

*Atropoides nummifer* Myotoxin I Absence 16.0 Cation-exchange chromatography on CM-Sephadex C-

*Atropoides nummifer* Myotoxin II Absence 13.7 8.7 P82950 Cation-exchange chromatography on CM-Sephadex C-

*Bothrocophias hyoprora* PhTX-I Presence 14.2 Reverse Phase chromatography on Bondapack® C-18

*Bothropoides insularis* SIII-SPVI Presence 15.0 Gel filtration chromatography on Sephadex G-150®

5.0 4.4

*Bothropoides insularis* Bi PLA2 Presence 13.9 8.6 Gel filtration chromatography on Superdex 75® column

*Bothropoides jararaca* BjPLA2 Presence 14.0 P81243 Ion-exchange chromatography on DEAE Sephacel®

*Bothropoides jararaca* PLA2 Presence 14.2 4.5 Q9PRZ0 Gel filtration on Sephacryl S-200® column and then

*Bothropoides pauloensis* BpPLA2 Presence 15.8 4.3 D0UGJ0 Cation-exchange chromatography on CM-Sepharose®

*Bothropoides pauloensis* BnSP-7 Absence 13.7 8.9 Q9IAT9 Cation-exchange chromatography on CM-Sepharose®

*Bothrops alternatus* BA SpII RP4 Presence 14.1 4.8 P86456 Gel filtration chromatography Sephadex G-75® column

*Bothrops alternatus* BaTX Absence 13.8 8.6 P86453 Gel filtration chromatography on Superdex 75® column

8.1- 8.3 8.1- 8.3 8.1- 8.3 8.1- 8.3 4.6

*Bothrops asper* Myotoxin I Presence 10.7 nd Ion-exchange chromatography on CM-Sephadex C-25®

*Bothrops asper* Myotoxin II Absence 13.3 nd P24605 Ion-exchange chromatography on CM-Sephadex C-25®

*Bothrops asper* Myotoxin III Presence 13.9 >9.5 P20472 Ion-exchange chromatography on CM-Sephadex C-25®

*Bothrops asper* BaspPLA 2-II Presence 14.2 4.9 P86389 Ion-exchange on CM-Sephadex C-25® followed by

*Bothrops alternatus* PLA2 Presence 15.0 5.0 Gel filtration chromatography on Sephadex G-50®

Access Number (Uniprot)

PLA2 Presence 14.0 Ion-exchange chromatography on DEAE-Cellulose®

MT1 Absence 14.0 9.0 49121 Anion-exchange chromatography on Waters DEAE-

Miotoxina II Presence 15.0 >9.5 P80963 Ion-exchange chromatography on CM-Sephadex®

Q8QG87 P84397

ACL-I Presence 14.0 Gel filtration chromatography on Superdex-200® column

column.

FF® column.

25® column.

25® column.

25® column.

column.

column.

column.

C8 column.

µ-Bondapack® C18 column.

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


venoms, which can be observed in the chromatograms shown in Figure 3.

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

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


Purification of Phospholipases A2 from American Snake Venoms 9

column, followed by reverse phase chromatography on

Reverse phase chromatography on µ-Bondapack® C18

Reverse phase chromatography on µ-Bondapack® C18

followed by chromatography on Mono-Q® and finally ion-exchange chromatography followed by DEAE-

followed by reverse phase chromatography on µ-

followed by reverse phase chromatography on µ-

Reverse phase on Vydac® C8 column. [129]

Gel filtration chromatography on Superdex 75® column, followed by reverse phase chromatography on µ-Bondapack® C-18 column and finally reverse phase

column followed by ion-exchange chromatography using MonoQ HR 5/5® column and finally reverse chromatography on Sephasil® C-18 column.

followed by reverse phase on Vydac® C18 column.

followed by ion-exchange chromatography on CM-

Gel filtration chromatography on Superdex G 75 HR® followed by reverse phase chromatography on Vydac®

Ion-exchange chromatography on Mono Q FF® column followed by reverse phase chromatography on Vydac® C4

column followed by reverse phase chromatography on µ-

Gel filtration chromatography on Sephadex G-75® column followed by ion-exchange chromatography on

Gel filtration chromatography on Sephacryl S-200® column, followed by reverse phase chromatography on C2 column and finally reverse phase chromatography on C18

[117]

[118]

[119]

[119]

[119]

[120]

[121]

[122]

[123]

[124]

[121]

[125]

[126]

[127]

[128]

[130]

[131]

[132]

[134]

[135]

[136]

[137]

[138]

[139]

Ion-exchange chromatography on CM-Sephadex®

Gel filtration chromatography on DEAE-cellulose®

reverse phase on µ-Bondapak® C-18 column.

column.

column.

column.

column.

column.

column.

column.

cellulose column.

CM-cellulose® column.

Bondapak® C18 column.

C18 column.

column.

chromatography.

chromatography.

Bondapack® C18.

cellulose® column.

Bondapack® C-18 column.

chromatography on C8 column.

PLA2 Presence 15.0 Gel filtration chromatography (pharmacia), followed by

F6a Presence 14.9 5.8 P0CAS2 Reverse phase chromatography on µ-Bondapack® C18 column.

Cdcum6 Presence 14.3 Nd P0CAS1 Gel filtration chromatography followed by reverse phase

PLA2A Presence 14.2 P86169 Gel filtration chromatography followed by reverse phase

CdtF16 Presence 14.8 P0CAS6 Gel filtration chromatography on Superdex 75® column,

Crotoxin B Presence 14.5 5.1 Gel filtration chromatography on Sephadex G75® column,

CdtF17 Presence 14.6 8.15 P0CAS7 Reverse phase chromatography on µ-Bondapack® C-18 column.

CdtF15 Presence 14.5 8.8 P0CAS5 Gel filtration chromatography on Superdex 75® column

P18998 P62023

P0C942 P0C943

P0C932 P0C933

*Porthidium nasutum* PnPLA2 Presence 15.8 4.6 Reverse phase chromatography on C18 column. [133] *Micrurus tener tener* MitTx-beta Presence 16.7 G9I930 Reverse phase chromatography on C18 Vydac® column

PLA2-1 Presence P25072 Gel filtration chromatography on Sephadex G-50®

P0CAS9 P0CAT0 P0CAT1

P81167

P21791 P21792

MiDCA1 Presence 15.5 8.0 Reverse phase chromatography on Sephasil Peptide® C18

P86805 P86806

P0CAS3 P0CAS4 Vydac® C8 column.

*Cerrophidion goodmani* Myotoxin I

*Crotalus atrox* PLA2–1

*Crotalus durissus cascavella* 

*Crotalus durissus collilineatus* 

*Crotalus durissus cumanensis* 

*Crotalus durissus cumanensis* 

*Crotalus durissus ruruima* 

*Crotalus durissus ruruima* 

*Crotalus durissus terrificus* 

*Crotalus durissus terrificus* 

*Crotalus durissus terrificus* 

*Crotalus durissus terrificus* 

*Crotalus scutulatus scutulatus* 

*Micrurus tener microgalbineus* 

*Lachesis muta* LmTX-I

*Lachesis muta* LM-PLA2-I

*Micrurus pyrrhocryptus* PLA2 A1

*Micrurus nigrocinctus* Nigroxin A

*Micrurus nigrocinctus* PLA2-1

*Micrurus dumerilli carinicauda* 

Myotoxin II

PLA2–2

Cdc-9 Cdc-10

Cdr-12 Cdr-13

MTX-a MTX-b

LmTX-II

LM-PLA2-II

PLA2 B1 PLA2 D5 PLA2 D6

Nigroxin B

PLA2-2 PLA2-3 Presence Absence

Absence Presence

Presence Presence

Presence Presence

 14.5 14.4

> 14.2 14.1

> 17.0 18.0

Presence Presence

Presence Presence

Presence Presence Presence Presence

Presence Presence

Presence Presence Presence 14.3 13.4

15.3 15.5

14.1 14.2

14.3 14.2 8.2 8.9

*Cerrophidion goodmani* GodMT-II Absence 13.7 Ion-exchange chromatography on CM-Sephadex®

4.6 8.6

*Crotalus atrox* Cax-K49 Absence 13.8 Q81VZ7 Gel filtration chromatography on DEAE-cellulose®

8.25 8.4

8.1 8.1

9.2 7.4

8.7 8.6

4.7 5.4

*Lachesis stenophys* LSPA-1 Presence 13.8 nd P84651 Gel filtration chromatography on Sephacryl S-200®

P0CAS8

P81166

P21790

*Cerrophidion goodmani* Pgo K49 Absence 13.8 Gel filtration chromatography on Sephadex G-75 HR®

**Table 1.** PLA2s isolated from American snake venoms and respective chromatographic methods used.

Some authors have proposed changes to the methodology described above. Spencer et al. [31] described the purification of BthTX-I with the use of Resourse S® (methyl-sulphonate


Presence Presence

Presence Absence

Absence Presence

Absence Presence

Presence Presence Presence Presence

Presence Presence Presence

Absence Presence

Presence Presence

Absence Absence 15.0 15.0

14.0 14.0

13.9 13.6

13.0 13.0

15.0 15.0 15.0 15.0

15.0 13.0 18.0

14.0 14.0

13.8 13.8

14.6 14.6 9.1 6.9

8.7 8.4

*Bothrops atrox* Myotoxin I Absence 13.8 8.9 Q6JK69 Ion-exchange chromatography on Carboximetil-Sephadex

*Bothrops erythromelas* BE-I-PLA2 Presence 13.6 Gel filtration chromatography on Superdex 75® followed

*Bothrops jararacussu* BJ IV Presence 15.0 P0CAR8 Ion-exchange chromatography on Protein Pack SP 5PW®

*Bothrops jararacussu* BthA-I-PLA2 Presence 13.7 4.5 Q8AXY1 Ion-exchange chromatography on CM-Sepharose®

5.3 5.3 5.3 5.3

5.3 5.3 5.3

*Bothrops leucurus* Bl-PLA2 Presence 15.0 5.4 P0DJ62 Ion-exchange chromatography on CM- Sepharose®

 P86974 P86975

 P86803 P86804

*Bothrops marajoensis* BmarPLA2 Absence 14.0 nd P0DI92 Ion-exchange chromatography on Protein Pack SP 5PW®,

*Bothrops marajoensis* Bmaj-9 Presence 13.7 8.5 B3A0N3 Reverse phase chromatography on µ-Bondapack® C18

*Bothrops moojeni* BthA-I Presence 13.6 5.2 G3DT18 Ion-exchange on CM-Sepharose® column, followed by

*Bothrops moojeni* MjTX-II Absence 14.0 8.2 Q9I834 Ion-exchange chromatography on CM-Sepharose®

*Bothrops moojeni* BmooTX-I Presence 15.0 4.2 Ion-exchange on DEAE-Sepharose®, gel filtration on

*Bothrops moojeni* BmTX-I Presence 14.2 7.8 P0C8M1 Reverse phase chromatography on µ-Bondapack® C18

*Bothrops moojeni* BmooMtx Absence 16.5 Ion-exchange chromatography on DEAE-Sephacel®

*Bothrops pirajai* Piratoxin-I Absence 13.8 8.3 P58399 Gel filtration chromatography on Sephadex G-75®

*Bothrops pirajai* Bpir-I PLA2 Presence 14.5 C9DPL5 Ion-exchange chromatography on CM- Sepharose FF®

*Bothrops pirajai* Piratoxin -II Absence 13.7 9.0 P82287 Gel filtration chromatography on Sephadex G-75®

8.2 Q90249 P45881

 Gel filtration chromatography on Sephacryl S-100 HR® column followed by reverse phase on C4 column.

column and then re-chromatographed on the same column

C-25® followed by reverse phase chromatography on C8

Reverse phase chromatography on C18 column. [102]

by chromatography on monoQ® column, fractions being subjected to reverse phase chromatography on C4 column

column followed by reverse phase chromatography on µ-

column, followed by reverse phase chromatography on

column, followed by ion-exchange chromatography on SP-Sephadex C-25® column, and finally HPLC on C18

Gel filtration chromatography on Sephacryl S-200®, followed by ion-exchange on Q-Sepharose and then submitted to reverse phase chromatography on HPLC

Ion-exchange chromatography on Protein Pack SP 5PW®, followed by reverse phase chromatography on µ-

Gel filtration chromatography on Sephadex G-75®, followed by cation-exchange chromatography on SP-

Presence 13.5 Ion-exchange chromatography on CM-Sephadex C-25®

column.

column.

afterwards.

C18 column.

C18 column.

Vydac® C4.

column.

Bondapack® C18 column.

Sepharose® column.

C18 column.

Absence 13.4 8.2 P82114 Ion-exchange chromatography on CM-Sepharose® column.

column.

column.

Presence 13.8 P58464 Ion-exchange chromatography on semi-preparative u-

C18 column.

C25® column.

G-75® column.

Sephadex C25® column.

Gel filtration chromatography on Superdex -75XK®

column.

and same conditions.

Ion-exchange chromatography on CM-Sepharose®

Sephadex C-25® column.

Bondapack® C18 column.

Gel filtration chromatography on Sephadex G-75®

Reverse phase chromatography on Lichrosfera RP100®

column, followed by hydrophobic interaction chromatography on Phenyl-Sepharose® column.

followed by reverse phase chromatography.

hydrophobic interaction chromatography on Phenyl-

column, followed by reverse phase chromatography on

Sephadex G-75® column and hydrophobic interaction chromatography on Phenyl-Sepharose®.

column and then submitted to gel filtration on Sephadex

column, followed by ion-exchange chromatography on

column, followed by reverse phase chromatography on

column and ion-exchange chromatography on Sephadex

Bondapack® column, followed by ion-exchange chromatography on Protein Pack SP 5PW® column. [99]

[32]

[100]

[101]

[103]

[34]

[104]

[58]

[105]

[106]

[38]

[43]

[107]

[108]

[109]

[24]

[110]

[26]

[111]

[42]

[112]

[113]

[114]

[115]

[25]

[116]

*Bothrops atrox* BaPLA2I

*Bothrops atrox* Basic

*Bothrops brazili* MTX-I

*Bothrops brazili* BbTX-II

*Bothrops jararacussu* BthTX-I

*Bothrops jararacussu* SIIISPIIA

*Bothrops lanceolatus* PLA2-1

*Bothrops leucurus* BLK-PLA2

*Bothrops marajoensis* BmjeTX-I

*Bothrops moojeni* MjTX-III

*Bothrops moojeni* MjTX-I ou

*Bothrops pirajai* Piratoxin-III

BaPLA2 III

Myotoxin

MTX-II

BbTX-III

BthTX-II

SIIISPIIB SIIISPIIIA SIIISPIIIB

PLA2-2 PLA2-3

BLD-PLA2

BmjeTX-II

MjTX-IV

Miotoxina-I

ou MPIII 4R

**Table 1.** PLA2s isolated from American snake venoms and respective chromatographic methods used.

Some authors have proposed changes to the methodology described above. Spencer et al. [31] described the purification of BthTX-I with the use of Resourse S® (methyl-sulphonate

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

Purification of Phospholipases A2 from American Snake Venoms 11

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

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

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

7.0.

separation.

**Figure 4.** Chromatographic profile of the *B.jararacussu* venom in CM-sepharose® column 1 ml (*Hitrap*) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1M (buffer B) in different pH conditions. **A**. pH 5.0 **B.** pH 6.0 **C.** pH 7.0 **D.** pH 8.0. Absorbance was read at 280 nm. Fractions numbered (1 to 8) indicate the fractions selected for SDS-PAGE analysis in order to confirm the presence of PLA2s (BthTx I e BthTx II).

class II (Figure 5).

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

**Figure 4.** Chromatographic profile of the *B.jararacussu* venom in CM-sepharose® column 1 ml (*Hitrap*) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1M (buffer B) in different pH conditions. **A**. pH 5.0 **B.** pH 6.0 **C.** pH 7.0 **D.** pH 8.0. Absorbance was read at 280 nm. Fractions numbered (1 to 8) indicate the fractions selected for SDS-PAGE analysis in order to

confirm the presence of PLA2s (BthTx I e BthTx II).

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

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

The combination of chromatographic techniques has also been used to purify these toxins. The association of the Ion-exchange chromatography and molecular exclusion has been one of the most recurrent in isolation and purification of phospholipases from bothropic venoms. Gel filtration chromatography is a technique based in particle size to obtain the separation. In this type of separation there is no physical or chemical interaction between the molecules of the analyte and the stationary phase, being currently used for separation of molecules with high molecular mass. The sample is introduced in a column, filled with a matrix constituted by small sized silica particles (5 to 10 µm) or a polymer containing a uniform net pores of which solvent and solute molecules diffuse. The retention time in the column depends on the effective size of the analyte molecules, the higher sized being the first ones to be eluted. Different from the higher molecules, the smaller penetrate the pores being retained and eluted later. Between the higher and lower molecules, there are the

intermediary sized molecules, whose penetration capacity in the pores depends on their diameter. In addition to that, this technique has also some very important characteristics, such as operational simplicity, physical chemical stability, inertia (absence of reactivity and adsorptive properties) and versatility, since it allows the separation of small molecules with mass under 100 Da as well as extremely big molecules with various kDa.

Purification of Phospholipases A2 from American Snake Venoms 13

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%

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

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

and 36% acetonitrile) and apparent molecular mass of 14 kDa.

toxins with a satisfactory purity degree, as shown in Table 1.

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

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

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

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

12 Chromatography – The Most Versatile Method of Chemical Analysis

the mass profile of the class II PLA2s.

analysis and good reproducibility.

al. [34].

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

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

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

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

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

mass under 100 Da as well as extremely big molecules with various kDa.

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.

Purification of Phospholipases A2 from American Snake Venoms 15

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.* 

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. jar*aracussu 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

Different authors used substrate analogous or reversible inhibitors coupled to the chromatographic resin. Rock and Snyder [50] were the first ones to use phospholipid analogous to build a bioaffinity matrix [Rac-1-(9-carboxy)-nonil-2-exadecilglycero-3 phosphocoline]. In addition to them, Dijkman [51] described the synthesis of an analogous of acylamino phospholipid[(R)-1-deoxy-1-thio-(ω-carboxy-undecyl)-2-deoxy- (ndecanoylamino)-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

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

from horse pancreas, and venoms from *Naja melanonleuca* and *Crotallus adamanteus*.

*jararacussu* venom and BthTX-I.

composed by lectin and BthTX-I.

**3. Characterization** 

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

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

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

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

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

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

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

## **3. Characterization**

14 Chromatography – The Most Versatile Method of Chemical Analysis

process.

and endothelial cells.

damaging effects on the cytoplasmic membrane.

be controlled altering the salt or reducing its concentration.

proteins that have surface hydrophobic domains. Hence, elution and protein separations can

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

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

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

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

mass of approximately 15 kDa) was obtained at the end of the process.

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.

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. 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

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

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

trichloroacetic acid for 30 minutes, followed by silver staining.

bidimensional electrophoresis.

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

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

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

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

16 Chromatography – The Most Versatile Method of Chemical Analysis

by the differences related to their mass

afterwards by mass spectrometry.

procedure (SDS-PAGE) as a protein characterization step.

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

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

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

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

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

Peptide Mass Fingerprint and the search for sequence similarity in data banks.

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

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

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

Purification of Phospholipases A2 from American Snake Venoms 19

**Figure 6.** Electrophoretic profile in 2D-PAGE 10%, 13 cm strip pH 3-10 non-linear of proteins from crude venom from *Bothrops moojeni*. Molecular weight (MW) –*Color Plus Prestained Protein Marker –* 

example of the practical application of this methodology can be seen in Figure 6.

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

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 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

*Broad Range* (7-175 kDa) (P7709S). Coomassie G-250.

**4. Functional characterization** 

the purification procedures, monitoring its presence.

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

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

**Figure 6.** Electrophoretic profile in 2D-PAGE 10%, 13 cm strip pH 3-10 non-linear of proteins from crude venom from *Bothrops moojeni*. Molecular weight (MW) –*Color Plus Prestained Protein Marker – Broad Range* (7-175 kDa) (P7709S). Coomassie G-250.

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