Journal of Chromatography B 1146 (2020) 122070

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Use of a phosphopeptide as a ligand to purify phospholipase A2 from the T

venom of Crotalus durisuss terrificus by affinity chromatography
Soledad L. Saavedraa,b,1, Gerardo Acostad,e,1, Lucía Ávilac, Silvana L. Giudicessia,b,
Silvia A. Camperia,b, Fernando Albericiod,e,f, Osvaldo Casconea,b,c, María C. Martínez Cerona,b,
⁎

a
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología, Junín 956, 1113 Buenos Aires, Argentina
b
CONICET-Universidad de Buenos Aires, Instituto de Nanobiotecnología (NANOBIOTEC), Junín 956, 1113 Buenos Aires, Argentina
c
Instituto Nacional de Producción de Biológicos, ANLIS Malbrán, Av. Vélez Sársfield 563, 1282 Buenos Aires, Argentina
d
Department of Organic Chemistry, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain
e
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, University of Barcelona, 08028 Barcelona, Spain
f
School of Chemistry & Physics, University of Kwazulu-Natal, Durban 4001, South Africa

ARTICLE INFO ABSTRACT

Keywords: The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with inter-
Snake venom esting pharmacological applications. Of these toxins, the phospholipase A2 (PLA2) subunit of crotoxin (Ctx) has
Phospholipase A2 been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the
Crotoxin purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought
Crotamine
to use a phosphopeptide (P-Lys) as a ligand for PLA2 purification. P-Lys was synthesized in solid phase on Rink-
Affinity chromatography
Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under
the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA2 activity.
Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA2
(14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA2 and its multimers. The final
product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the
isolation of crotamine.

1. Introduction aureus), at different inhibitory doses has been tested [10–13].

Snake venom contains many toxins that have a wide range of effects
on prey. However, venom is also a rich source of molecules with
pharmacological applications. In this regard, the potential applications
of Crotalus durissus terrificus (Cdt) venom has been widely studied. The
main toxin found in this venom is crotoxin (Ctx), a β-neurotoxin that
accounts for almost 50% of the dry weight of the venom [1–3]. Ctx is a
non-covalently bonded heterodimer formed by an enzymatically in-
active subunit (crotapotin, crotoxin A or CA) and a phospholipase A2
(PLA2, crotoxin B or CB) [4–6]. The potential use of the PLA2 subunit as
an antiviral agent, especially against viruses of the Flavivirus genus
such as Dengue and Yellow Fever, has been reported [7–9]. Further-
more, the capacity of PLA2 as an antibacterial agent against both Gram-
negative (Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, Bur-
kholderia pseudomallei) and Gram-positive bacteria (Staphylococcus
The production of pure PLA2 is of great interest. The methodologies
currently used to purify this venom protein are suitable for the la-
boratory scale but difficult to implement for an industrial scale. For
example, Faure and Bon [14] purified the crotoxin from Cdt venom
using Sephadex G-75 molecular exclusion chromatography (SEC), then
used FPLC on an anion exchange column, Mono-Q HR16 / 10 to se-
parate the different isoforms of crotoxin. To isolate the crotoxin sub-
units, CA and PLA2, they used ion-exchange chromatography (IEC),
DEAE-cellulose DE-52 columns, in the presence of 6 M urea. Muller
et al. [7] also used a SEC Sephadex G-75 as a first step and then after
dialysis and lyophilization, they incubated the sample with 6 M Urea,
ultrafiltrated and separated CA from PLA2 using an IEC DEAE-Se-
pharose chromatography to finally re-dialyze and lyophilize the pro-
tein. The two cases exemplified here consist of numerous steps to se-
parate the PLA2 from its chaperone currently used. Each purification

Corresponding author at: Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología and CONICET-Universidad de Buenos Aires,
⁎

Instituto de Nanobiotecnología (NANOBIOTEC), Junín 956, 1113 Buenos Aires, Argentina.

E-mail address: camartinez@ffyb.uba.ar (M.C. Martínez Ceron).
1
Both authors collaborated equally.

https://doi.org/10.1016/j.jchromb.2020.122070
Received 18 October 2019; Received in revised form 6 March 2020; Accepted 14 March 2020
Available online 16 March 2020
1570-0232/ © 2020 Elsevier B.V. All rights reserved.
S.L. Saavedra, et al.
step leads to a decrease of the overall yield [15] and, of course, an
increase in the production cost. On the other hand, the method de-
scribed herein allows the purification of PLA2 in just a single step.
Peptides have proven useful ligands for the affinity purification of
numerous molecules, such as antibodies, blood factors, milk proteins,
toxins, and other proteins. They can be synthesized at low cost under
GMP standards [15–21] and they are much more stable than proteins,
as they do not require a specific tertiary structure to fulfill their func-
tion. Furthermore, peptides can be easily modified to increase their
chemical stability and resistance to proteases, such as those present in
the Cdt venom.
In a previous study, we synthesized and screened a peptide com-
binatorial library [22], and identified two sequences that initially ap-
peared to purify PLA2 but were found to bind to Ctx instead. We
therefore decided to synthesize a phosphopeptide (P-Lys) to mimic the
phospholipid structure [23] —the natural enzymatic substrate of PLA2
and use it as a chromatographic ligand to purify the enzyme from Cdt
venom in a single step.
2. Materials and methods
2.1. Materials
Fluorenylmethoxycarbonyl(Fmoc)-D-Proline, Fmoc-O-(benzylpho-
spho)-L-serine, and OxymaPure (2-cyano-2-(hydroxyimino)acetate)
were from Iris Biotech GmbH (Marktredwitz, Germany). Trifluoroacetic
acid (TFA), N,N'-diisopropylcarbodiimide (DIPCDI), N,N-diisopropy-
lethylamine (DIPEA), bovine serum albumin (BSA), and triisopropylsi-
lane (TIS) were supplied by Sigma-Aldrich (Saint Louis, MO, USA).
Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluoropho-
sphate (PyBOP), Rink-amide-ChemMatrix resin (100–200 mesh,
0.45 meq/mg), and other Fmoc-protected amino acids were purchased
from Peptides International Inc. (Louisville, KY, USA). Crotalus durissus
terrificus venom was from the Instituto Nacional de Producción de
Biológicos, ANLIS Malbrán (Buenos Aires, Argentina). N-
Hydroxysuccinimide EZ-Link® Dry Pierce™ NHS-Activated Agarose and
Low Molecular Weight (LMW) Protein Marker PageRuler were provided
by Thermo Fisher Scientific (Rockford, IL, USA). 4-nitro-3-(octanoy-
loxy)benzoic acid (4N3OBA) reagent was from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Bradford reagent (Bio-Rad
Protein Assay Dye Reagent) was purchased from Bio-Rad Laboratories
(Philadelphia, PA, U.S.A.). All other reagents were AR grade.
3. Methods
3.1. Phosphopeptide synthesis
The peptide Ac-Phe-Phe-Val-Arg-D-Pro-pSer-Glu-Val-Phe-Phe-Lys-
NH2 (P-Lys) (Fig. S1) was synthesized on Rink-Amide-ChemMatrix resin
at room temperature, using the Fmoc/tBu strategy [24]. Before starting
the coupling, the resin was washed first with DMF (2 × 5 min) and then
with CH2Cl2. The addition of all the residues, except Arg, was accom-
plished with Fmoc protected amino acid (3 eq), OxymaPure (3 eq) and
DIPCDI (3 eq) in DMF and by incubating the mixture for 60 min at room
temperature. Protected Arg was introduced after the D-Pro using 3 eq of
PyBOP and 3 eq of DIPEA in a microwave oven (2 min at 20% of
power). LiCl (0.4 M) in DMF was added to the mixture to make the D-
Pro more accessible for the incorporation of the following amino acids.
To confirm the success of the coupling of each Fmoc amino acid, the
Kaiser test was performed [25]. A mix of Piperidine:DMF (20:80)
(2 × 10 min) was used to remove the Fmoc protecting group after each
coupling. A washing step using DMF and CH2Cl2 (5 × 1 min) was
performed between couplings and Fmoc removal. Acetylation of the N-
terminus amino acid was achieved by adding 10 eq of Ac2O and 10 eq
of DIEA in CH2Cl2 anhydrous. Finally, to remove the side chain pro-
tecting groups and separate the peptide from the resin, a TFA/TIS/H2O
2
Journal of Chromatography B 1146 (2020) 122070
(95:2.5:2.5) treatment was performed for 2 hours. The peptide was
recovered and precipitated in a flask with cold diethyl ether and then
centrifuged at 4 °C for 10 min at 10,000 rpm. Afterwards, the peptide
mass was dissolved with distilled water and lyophilized.
3.2. Phosphopeptide analysis and purification
The phosphopeptide was characterized by HPLC-MS-ESI on a
Waters PDA Photodiode array detector 2998 System with a XBridge
BEH130 k reverse-phase C18 column (4.6 × 100 mm, 3.5 µm) using a
linear gradient from 100% A (=0.045% TFA in water) to 80% B
(=0.036% TFA in MeCN) in 8 min, at a flow rate of 1.0 mL/min for
14 min. A 10.0 µL aliquot of peptide solution was injected into the
column. Mass spectra were acquired in the MS reflectron positive-ion
mode.
3.3. Synthesis of the phosphopeptidyl-agarose affinity matrix
The peptide was immobilized on agarose as described by Martínez-
Ceron et al. [26].
3.4. Peptide coupling measurement
The amount of immobilized peptide in the matrix was determined as
the difference between the concentration before and after the im-
mobilization step. These concentrations were determined by HPLC
following Martínez-Ceron et al. [26].
3.5. Affinity chromatography
A sample of 100 μL of a 1/20 dilution of venom (11.91 mg/mL) in
adsorption buffer was loaded to the column (0.5 × 5 cm) filled with the
agarose-peptide matrix. The column was washed until absorbance at
280 nm reached its initial value with equilibrating buffer. Two equili-
brating buffers were assayed: 1) 0.05 M Tris/HCl, pH 8.0, 0.01 M CaCl2,
0.1 M NaCl; and 2) 0.1 M sodium acetate, pH 4.5, 0.025 M EDTA, 0.5 M
NaCl. Once the sample was loaded, the resin was left agitating in batch
for 30 min. The following eluents were assayed: 1) PBS, pH 7.4; 2)
0.05 M Tris/HCl, 0.01 M EDTA, pH 8.5; 3) 0.01 M Tris/HCl, pH 8.0, 5 M
LiCl; 4) 0.01 M Tris/HCl, pH 8.0, 2 M NaCl; and 5) 0.05 M NaOH.
3.6. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) and Tricine-SDS-PAGE
A 15% SDS-PAGE under reductive conditions was performed as
described by Laemmli [27] and then stained with silver as described by
Aboulaich [28]. A 16% Tricine-SDS-PAGE was carried out as described
by Schägger [29] and then stained with Coomassie G-250 for mass
spectrometry analysis.
3.7. Protein quantification
The concentration of the protein fractions collected from the chro-
matographic process from the chromatographic column was measured
by the Bradford method [30] using a 96-well plate and a FlexStation® 3
Multi-Mode Microplate Reader (Molecular Devices, San José, CA, USA)
at 595 nm. A BSA standard curve was used.
3.8. Enzyme activity assay
To measure phospholipase activity, a modification of the method
described by Petrovic et al. [31] was used. A 0.01% solution of 4N3OBA
(Santa Cruz Biotechnology, USA) in MeCN was prepared, and fractions
of 0.1 mL were loaded into vials, lyophilized and stored at −20 °C.
Before use, 0.5 mL of 0.01 M Tris-HCl, pH 8.0, 0.01 M CaCl2, 0.1 M
NaCl buffer was loaded per vial. To measure activity, 0.18 mL of the
S.L. Saavedra, et al.
4N3OBA solution and 0.02 mL of samples or water were loaded in a 96-
well plate. The activity was measured at 37° C every 5 min over 50 min
using a FlexStation® 3 Multi-Mode Microplate Reader (Molecular De-
vices, USA) apparatus at 425 and 600 nm for correction. All the samples
were analyzed in triplicate. The activity, measured in EU (Enzymatic
Units) per mL, was calculated using Equation (1).
Eq. (1). Where: “A” is absorbance and “OD” is optical density, ob-
tained after the absorbance correction. The correction factor of 0.07862
is the product concentration (µmoles in 0.2 mL, final reaction volume)
that generates an absorbance of 1.0 at 425 nm, under the experimental
conditions.
µmol OD 425 µmol
PLA2 activity = (A425 A600) × 0.07862
min×mL min OD 425
1 1
×
sample vol mL
3.9. Protein identification
Proteins from the Tricine-SDS-PAGE gel stained with colloidal
Coomassie G-250 were identified by peptide mapping analysis. The
selected bands were cut and placed on vials, reduced with 0.02 M di-
thiothreitol (DTT) at 56 °C for 45 min, alkylated with iodoacetamide for
45 min in darkness and digested with trypsin at 37 °C overnight. The
peptides were extracted with MeCN and lyophilized. Finally, the sam-
ples were resuspended in 0.01 mL of 0.1% formic acid and desalted
with a C18 Zip Tip. nanoHPLC electrospray ionization mass spectro-
metry (HPLC-ESI-MS) with Orbitrap technology (ThermoScientific,
EASY-nLC 1000 model) with a C18 column (2 µm, 75 µm × 150 mm)
was performed at 35° C. The analysis was performed using a linear
gradient starting from a mobile phase 95% of A to another 95% of B
within 120 min, where the mobile phase A = H2O (0.1% formic acid)
and B = MeCN (0.1% formic acid). The flow used was 300 nL / min and
the injected sample volume was 2 µL. The capillary and cone voltage of
the mass spectrometry was 3.5 kV and 35 V, respectively. Data were
analyzed with the program Proteome Discoverer v1.4 (Thermo
Scientific), comparing against the proteome database of Cdt venom.
4. Results and discussion
4.1. Peptide design and synthesis
To design the peptide, the phospholipids as the PLA2 targets were
considered. Pellach et al. [23] designed a peptide resembling a phos-
pholipid: a 10-amino acid sequence adopting a β-sheet conformation to
generate a two-hydrophobic tail structure with a phosphorylated serine
in the middle to act as the polar “head”. To help stabilize the hairpin-
like structure, they included oppositely charged Lys and Glu flanking
the polar head and hydrophobic amino acids on both ends of the se-
quence. We based our design on that structure but adding several
modifications. In this regard, a Lys residue was incorporated at the C-
terminal to allow peptide coupling to an agarose support through the
amine side chain. To avoid a second point of attachment, the Lys in the
original sequence was replaced by Arg to maintain the original ionic
charge. The final sequence was, therefore, Ac-Phe-Phe-Val-Arg-D-Pro-
pSer-Glu-Val-Phe-Phe-Lys-NH2 (P-Lys). The peptide was synthesized on
Rink-Amide-ChemMatrix resin by Fmoc/tBu chemistry, achieving
49.7 mg of peptide with a purity of 96% after HPLC purification (Figs.
S2 and S3). The molecular mass obtained by mass spectrometry
([M + H]+ = 1524.8 u.m.a.) was equivalent to that calculated theo-
retically (MW = 1523.69 u.m.a.). The peptide amide was synthesized
to prevent peptide polymerization during coupling.
Journal of Chromatography B 1146 (2020) 122070
0,30
0,25
Absorbance at 280 nm
0,20
0,15
0,10
0,05
0,00
0 5 10 15 20
(1) Volume (mL)
Fig. 1. Cdt venom chromatography with P-Lys-agarose matrix. A venom dilu-
tion (100 µL of 11.8 mg/mL) in buffer 0.01 M Tris/HCl, pH 8.0, 0.01 M CaCl2,
0.1 M NaCl was loaded on the P-Lys-agarose matrix column (0.5 × 5 cm). The
column was washed with equilibrating buffer at a flow rate of 0.25 mL/min
until absorbance at 280 nm reached baseline value. The best result for the P-
Lys-agarose matrix was achieved using 0.1 M sodium acetate, pH 4.5, 0.025 M
EDTA, 0.5 M NaCl as the elution buffer 1 (dash arrow) and 0.05 M NaOH as
elution buffer 2 (solid arrow).
4.2. Generation of the phosphopeptidyl-resin
The phosphopeptide was immobilized on NHS-agarose resin
through the amine side chain of Lys. The concentration of the im-
mobilized ligand was measured indirectly from the uncoupled amount
of peptide present in the recovered reaction mixture and washings after
the immobilization reaction. A ligand density of 19 μmol of peptide per
mL of matrix was achieved.
4.3. Affinity chromatography (AC)
Several buffers were tested to determine optimal chromatographic
conditions. Proteins were recovered in both fractions when using
0.05 M Tris/HCl, 0.01 M CaCl2, 0.1 M NaCl, pH 8.0 as adsorption
buffer, and 0.1 M sodium acetate, 0.025 M EDTA, 0.5 M NaCl, pH 4.5 as
elution buffer, however no PLA2 activity was detected in either. A
second elution step using 0.05 M NaOH allowed the recovery of a
protein fraction with PLA2 activity. The chromatogram obtained
showed that 70% of the total protein of the venom was adsorbed and
later recovered in both elution steps (Fig. 1). No protein with the ex-
pected PLA2 molecular weight was present in the pass-through or in the
eluate 1 fractions, as determined by SDS-PAGE (Fig. 2). However, in the
eluate 2 fraction, three bands were present, one of approximately
14 kDa.
Mass spectrometry of the four selected bands from both eluates
(Fig. 2B) revealed that crotamine (Ctm)—a low molecular weight pro-
tein present in some venoms of Cdt—was recovered in eluate 1 and
PLA2 was recovered in eluate 2. The two proteins of higher molecular
weight present in eluate 2 were also identified as PLA2, which means
the enzyme formed multimers once separated from crotapotin as de-
scribed by Marchi-Salvador et al. [32] and Faure et al. [33]. The pep-
tides obtained from the digestion and LC-MS/MS analysis presented
high scores and a high coverage rate (Table S1 and S2) for all the
proteins analyzed.
Ctm has been reported to exert a potent analgesic effect [34] and to
show activity against several fungi of Candida spp., in particular C.
albicans [35]. To understand why the matrix with the phosphopeptide
immobilized also adsorbs Ctm, isoelectric point (pI) value of the peptide
was determined using the application designed by Innovagen (https://
pepcalc.com/). For P-Lys, the pI value calculated was 8.13. Ctm is a
3
S.L. Saavedra, et al.
0,600
0,500
Absorbance at 280 nm
0,400
0,300
0,200
0,100
0,000
0 2 4 6 8 10
Volume (mL)
Fig. 3. New condition of Cdt venom chromatography with a P-Lys-agarose
column. A venom dilution (100 µL of 11.8 mg/mL) in buffer 0.1 M sodium
acetate, pH 4.5, 0.025 mM EDTA, 0.5 M NaCl was loaded on the P-Lys-agarose
column The column was washed with equilibrating buffer at a flow rate of
0.25 mL/min until the absorbance at 280 nm reached baseline value. The best
result for the P-Lys-agarose matrix was achieved by eluting with 0.05 M NaOH.
The arrow indicates the buffer change.
highly positive charged protein, as reported by Kerkis et al. [35], with a
pI value of 9.54. Therefore, the interaction between P-Lys and Ctm
under our work conditions could be explained as being mainly of an
electrostatic nature. For the affinity chromatography, 0.05 M Tris/HCl,
pH 8.0, 0.01 M CaCl2, 0.1 M NaCl was used as adsorption buffer. Under
these conditions, the matrix was practically neutral and Ctm had a
positive charge, therefore, the interaction is mainly hydrophobic. With
0.1 M sodium acetate, 0.025 M EDTA, 0.5 M NaCl, pH 4.5 as elution
buffer, Ctm could be released from the matrix because both the matrix
and the protein acquire positive charge.
The CA subunit of crotoxin has the function of carrying PLA2 to a
specific receptor anchored in the presynaptic membrane. When cro-
toxin reaches the target, PLA2 binds to the membrane and CA is re-
leased [36]. Probably, during the chromatographic process described
here, Ctx interacts with the peptide attached to the agarose matrix,
PLA2 recognizes a similar structure to phospholipids (its enzymatic
target), binds to it and releases the CA subunit. Once separated, the CA
subunit is lost in the passthrough. CA is a small protein (8–9 kDa)
[14,37] comprised by 3 chains [38] linked by disulfide bridges. Under
4
Journal of Chromatography B 1146 (2020) 122070
Fig. 2. Analysis of the chromatographic fractions
by SDS-PAGE. A. Silver-stained SDS-PAGE gel
(15%) of Cdt venom chromatography using a P-Lys-
agarose column (0.5 × 5 cm). Lane 1, molecular
weight marker; lane 2, Cdt venom; lane 3, pass-
through fraction; lane 4, eluted fraction 1; and lane
5, eluted fraction 2. B. Colloidal Coomassie Tricine-
SDS-PAGE gel (16%) of Cdt venom chromatography
using a P-Lys-agarose column (0.5 × 5 cm). Lane 1,
pass-through fraction; lane 2, eluted fraction 1; lane
3, eluted fraction 2; lane 4, Cdt venom; and lane 5,
molecular weight marker. Squares show the sam-
ples selected for identification analysis by mass
spectrometry. The SDS-PAGE and mass spectro-
metry showed that the band corresponding to Ctm
(4.5 kDa) appeared only in elution fraction 1 and
the band of PLA2 (14 kDa) appeared only in elution
fraction 2.
Fig. 4. Analysis of the chromatographic fractions by SDS-PAGE. Silver-stained
Tricine-SDS-PAGE gel (15%) of Cdt venom chromatography in a P-Lys-agarose
column (0.5 × 5 cm). Lane 1, molecular weight marker; lane 2, Cdt venom, lane
3, pass-through fraction; and lane 4, eluted fraction. The band corresponding to
PLA2 (14 kDa) appeared only in the elution fraction.
the reducing conditions of the SDS-PAGE, the chains are separated and
cannot be visualized in the gel because of their low molecular weight.
In this context, the purification conditions used herein are suitable
to recover two potentially useful proteins rather than only one.
4.4. Optimized AC for PLA2 recovery
To simplify the PLA2 purification process, 0.1 M sodium acetate,
0.025 M EDTA, 0.5 M NaCl, pH 4.5, as the new adsorption buffer, and
various eluents were assayed to recover PLA2. Only 0.05 M NaOH was
found to be effective for enzyme recovery. Under these conditions,
adsorption of the total venom proteins reached 39% (Fig. 3). The SDS-
PAGE revealed that, in this case, Ctm was lost in the wash fraction and
only PLA2 was recovered after elution with 0.05 M NaOH (Fig. 4). These
conditions allowed us to obtain high purity PLA2 (90%), as determined
S.L. Saavedra, et al.
Table 1
Purification of PLA2 from Cdt venom with P-Lys-agarose matrix.
Volume (mL) Total Protein (mg)
Cdt venom (11.8 mg/mL) 0.2 2.38
Pass-through 8 1.5
Elution fraction 5.0 0.39
a
Purity (%) = (Amount of target protein/Amount of total protein) × 100.
b
Specific activity = total PLA2 activity/Amount of total protein.
by the scanning of the SDS-PAGE using the ImageJ v 1.50i (National
Institutes of Health) program.
The protein concentration and the enzymatic activity of the crude,
passthrough and elution fractions are shown in Table 1. It was not
possible to calculate the yield (Y%) and the purification factor because
the enzymatic activity of the free PLA2 exceeds (in some cases up to 4
times) the phospholipase activity in the crotoxin complex [32,33,39],
thereby giving meaningless numerical values. In addition, the use of
0.05 M NaOH as eluent did not affect enzymatic activity.
5. Conclusions
The phosphopeptide designed herein showed the capacity to purify
PLA2 in a single chromatographic step. The protein was obtained with
more than 90% purity starting from Cdt venom, which is a complex
sample, and it remained enzymatically active despite the conditions
used to recover it. Although multimeric forms of PLA2 would not pre-
sent the desired level of enzymatic activity and can be considered im-
purities, they can be removed by a subsequent polishing step.
In addition to PLA2, using the same matrix but slightly changing
chromatographic conditions, we have been able to purify Ctm [40], a
protein with interesting pharmacological properties that is not present
in all Cdt venoms.
In order to assess the possibility of using P-Lys to purify PLA2s from
other sources, tests with venom of Bothrops alternatus and B. neuwiedii
were also performed but the PLA2s could not be purified under the
conditions assayed. Both were recovered in the passthrough (data not
shown). Therefore, we cannot ensure that P-Lys is a species-specific
matrix. Possibly these PLA2s could not be purified by this matrix be-
cause of the small differences that exist between enzyme recognition
sites. It would be interesting to evaluate it through simulation tools by
molecular dynamics or crystallography, for example, but it is beyond
the scope of this work, as well as test this matrix with other samples to
evaluate its selectivity.
Although more research is needed before these proteins can be used
as biopharmaceuticals, PLA2 and Ctm could be used as templates for the
research and development of new therapeutic agents to treat cancer,
and also as new sources of antiviral, antimicrobial or analgesic com-
pounds as well.
CRediT authorship contribution statement
Soledad L. Saavedra: Conceptualization, Methodology,
Investigation, Writing - original draft, Writing - review & editing,
Visualization. Gerardo Acosta: Conceptualization, Methodology,
Investigation, Writing - review & editing. Lucía Ávila: Methodology,
Writing - review & editing. Silvana L. Giudicessi: Conceptualization,
Methodology, Writing - original draft, Writing - review & editing. Silvia
A. Camperi: Conceptualization, Methodology, Resources, Writing -
review & editing, Supervision, Project administration, Funding acqui-
sition. Fernando Albericio: Methodology, Resources, Writing - review
& editing, Supervision, Funding acquisition. Osvaldo Cascone:
Conceptualization, Methodology, Resources, Writing - review & editing,
Supervision, Project administration, Funding acquisition. María C.
Martínez Ceron: Conceptualization, Methodology, Investigation,
5
Journal of Chromatography B 1146 (2020) 122070
a b
Total Activity (IU) Purity (%) Specific activity (IU/mg)
0.12 39.0 0.05
0.04 – 0.023
0.26 90.0 0.66
Resources, Writing - original draft, Writing - review & editing,
Visualization, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
None.
Acknowledgements
This work was partially supported by the Universidad de Buenos
Aires (20020170100030BA), Argentina, the Ministerio de Ciencia,
Tecnología e Innovación Productiva de la República Argentina
(PICT‐2016‐0751), and the Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET PIP 11220130100119CO), Argentina.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jchromb.2020.122070.
References
[1] N. Gralén, T. Svedberg, The molecular weight of crotoxin, Biochem. J. 32 (1938)
1375–1377, https://doi.org/10.1042/bj0321375.
[2] S.P. Muller, V.A.O. Silva, A.V.P. Silvestrini, L.H. de Macedo, G.F. Caetano,
R.M. Reis, M.V. Mazzi, Crotoxin from Crotalus durissus terrificus venom: In vitro
cytotoxic activity of a heterodimeric phospholipase A2 on human cancer-derived
cell lines, Toxicon. 156 (2018) 13–22, https://doi.org/10.1016/J.TOXICON.2018.
10.306.
[3] K.H. Slotta, H. Fraenkel-Conrat, Two active proteins from rattlesnake venom [9],
Nature 142 (1938) 213, https://doi.org/10.1038/142213a0.
[4] G. Faure, V. Choumet, C. Bouchier, L. Camoin, J.-L. Guillaume, B. Monegier,
M. Vuilhorgne, C. Bon, The origin of the diversity of crotoxin isoforms in the venom
of Crotalus durissus terrificus, Eur. J. Biochem. 223 (1994) 161–164, https://doi.
org/10.1111/j.1432-1033.1994.tb18978.x.
[5] E. Habermann, H. Breithaupt, The crotoxin complex—an example of biochemical
and pharmacological protein complementation, Toxicon. 16 (1978) 19–30, https://
doi.org/10.1016/0041-0101(78)90056-9.
[6] S. Hernandez-Oliveira, M.H. Toyama, D.O. Toyama, S. Marangoni, S. Hyslop,
L. Rodrigues-Simioni, Biochemical, pharmacological and structural characterization
of a new PLA2 from Crotalus durissus terrificus (South American Rattlesnake)
venom, Protein J. 24 (2005) 233–242, https://doi.org/10.1007/s10930-005-
6718-z.
[7] V.D.M. Muller, R.R. Russo, A.C. Oliveira Cintra, M.A. Sartim, R. De Melo Alves-
Paiva, L.T.M. Figueiredo, S.V. Sampaio, V.H. Aquino, Crotoxin and phospholipases
A2 from Crotalus durissus terrificus showed antiviral activity against dengue and
yellow fever viruses, Toxicon. 59 (2012) 507–515, https://doi.org/10.1016/J.
TOXICON.2011.05.021.
[8] V.D. Muller, R.O. Soares, N.N. dos Santos, A.C. Trabuco, A.C. Cintra,
L.T. Figueiredo, A. Caliri, S.V. Sampaio, V.H. Aquino, Phospholipase A2 isolated
from the venom of Crotalus durissus terrificus inactivates dengue virus and other
enveloped viruses by disrupting the viral envelope, PLoS One 9 (2014) e112351,
https://doi.org/10.1371/journal.pone.0112351.
[9] R.R. Russo, N.N. dos Santos Júnior, A.C.O. Cintra, L.T.M. Figueiredo, S.V. Sampaio,
V.H. Aquino, Expression, purification and virucidal activity of two recombinant
isoforms of phospholipase A 2 from Crotalus durissus terrificus venom, Arch. Virol.
164 (2019) 1159–1171, https://doi.org/10.1007/s00705-019-04172-6.
[10] J.R. Almeida, A.L.V. Palacios, R.S.P. Patiño, B. Mendes, C.A.S. Teixeira, P. Gomes,
S.L. da Silva, Harnessing snake venom phospholipases A 2 to novel approaches for
overcoming antibiotic resistance, Drug Dev. Res. 80 (2019) 68–85, https://doi.org/
10.1002/ddr.21456.
[11] R. Perumal Samy, A. Pachiappan, P. Gopalakrishnakone, M.M. Thwin, Y.E. Hian,
V.T. Chow, H. Bow, J.T. Weng, In vitro antimicrobial activity of natural toxins and
animal venoms tested against Burkholderia pseudomallei, BMC Infect. Dis. 6 (2006)
100, https://doi.org/10.1186/1471-2334-6-100.
S.L. Saavedra, et al.
[12] R.P. Samy, P. Gopalakrishnakone, B.G. Stiles, K.S. Girish, S.N. Swamy,
M. Hemshekhar, K.S. Tan, E.G. Rowan, G. Sethi, V.T.K. Chow, Snake venom
phospholipases A(2): a novel tool against bacterial diseases, Curr. Med. Chem. 19
(2012) 6150–6162 (accessed June 25, 2019), http://www.ncbi.nlm.nih.gov/
pubmed/22963667.
[13] R.P. Samy, B.G. Stiles, O.L. Franco, G. Sethi, L.H.K. Lim, Animal venoms as anti-
microbial agents, Biochem. Pharmacol. 134 (2017) 127–138, https://doi.org/10.
1016/j.bcp.2017.03.005.
[14] G. Faure, C. Bon, Crotoxin, a phospholipase A2 neurotoxin from the South American
rattlesnake Crotalus durissus terrificus: purification of several isoforms and com-
parison of their molecular structure and of their biological activities, Biochemistry
27 (1988) 730–738, https://doi.org/10.1021/bi00402a036.
[15] R.P. Samy, M. Maung Thwin, B.G. Stiles, H. Bow, V.T.K. Chow,
P. Gopalakrishnakone, Therapeutic potential of peptides with neutralizing ability
towards the venom and toxin (CaTx-I) of Crotalus adamanteus, Curr. Top. Med.
Chem. 11 (2011) 2540–2555, https://doi.org/10.2174/156802611797633384.
[16] S.A. Camperi, S.L. Giudicessi, M.C. Martínez-Ceron, J.M. Gurevich-Messina,
S.L. Saavedra, G. Acosta, O. Cascone, R. Erra-Balsells, F. Albericio, Combinatorial
library screening coupled to mass spectrometry to identify valuable cyclic peptides,
Curr. Protoc. Chem. Biol. 8 (2016) 109–130, https://doi.org/10.1002/cpch.2.
[17] S.L. Saavedra, L. Avila, S.L. Giudicessi, F. Albericio, S.A. Camperi, O. Cascone,
M.C. Martinez-Ceron, Natural snake venom inhibitors and their pharmaceutical
uses: challenges and possibilities, Curr. Pharm. Des. 24 (2018) 1737–1747, https://
doi.org/10.2174/1381612824666180223172854.
[18] S.L. Saavedra, M.C. Martínez Ceron, S.L. Giudicessi, G. Forno, M.B. Bosco,
M.M. Marani, R. Erra-Balsells, F. Albericio, O. Cascone, S.A. Camperi, Single step
recombinant human growth hormone (rhGH) purification from milk by peptide
affinity chromatography, Biotechnol. Prog. 34 (2018) 999–1005, https://doi.org/
10.1002/btpr.2645.
[19] J.M. Gurevich Messina, S.L. Giudicessi, M.C. Martínez Ceron, N. Urtasun, G. Forno,
L. Mauro, O. Cascone, S.A. Camperi, Recombinant human follicle stimulating hor-
mone purification by a short peptide affinity chromatography, J. Pept. Sci. 24
(2018) e3128, https://doi.org/10.1002/psc.3128.
[20] N. Kruljec, P. Molek, V. Hodnik, G. Anderluh, T. Bratkovič, Development and
characterization of peptide ligands of immunoglobulin G Fc region, Bioconjug.
Chem. 29 (2018) 2763–2775, https://doi.org/10.1021/acs.bioconjchem.8b00395.
[21] T. Islam, A.D. Naik, Y. Hashimoto, S. Menegatti, R.G. Carbonell, Optimization of
sequence, display, and mode of operation of IgG-binding peptide ligands to develop
robust, high-capacity affinity adsorbents that afford high IgG product quality, Int. J.
Mol. Sci. 20 (2019), https://doi.org/10.3390/IJMS20010161.
[22] M.C. Martínez-Ceron, S.L. Saavedra, L. Ávila, S.L. Giudicessi, F. Albericio, S.A.
Camperi, O. Cascone, One-Bead-One-Peptide Library to Purify Crotalus durissus
terrificus Phosholipase A2, (2015). https://doi.org/10.17952/24APS.2015.137.
[23] M. Pellach, Y. Atsmon-Raz, E. Simonovsky, H. Gottlieb, G. Jacoby, R. Beck, L. Adler-
Abramovich, Y. Miller, E. Gazit, Spontaneous structural transition in phospholipid-
inspired aromatic phosphopeptide nanostructures, ACS Nano 9 (2015) 4085–4095,
https://doi.org/10.1021/acsnano.5b00133.
[24] W.C. Chan, P.D. White, Basic procedures, Fmoc Solid Phase Pept. Synth. 2000, pp.
41–76.
[25] E. Kaiser, R.L. Colescott, C.D. Bossinger, P.I. Cook, Color test for detection of free
terminal amino groups in the solid-phase synthesis of peptides, Anal Biochem. 34
(1970) 595–598 http://www.ncbi.nlm.nih.gov/pubmed/5443684.
Journal of Chromatography B 1146 (2020) 122070
[26] M.C. Martínez-Ceron, M.M. Marani, M. Taulés, M. Etcheverrigaray, F. Albericio,
O. Cascone, S.a. Camperi, Affinity chromatography based on a combinatorial
strategy for rerythropoietin purification, ACS Comb. Sci. 13 (2011) 251–258,
https://doi.org/10.1021/co1000663.
[27] U.K. Laemmli, Cleavage of Structural Proteins during the Assembly of the Head of
Bacteriophage T4, Nature 227 (1970) 680–685, https://doi.org/10.1038/
227680a0.
[28] N. Aboulaich, Silver Staining, Bio-Protocol. 1 (2011) e26, https://doi.org/10.
21769/BioProtoc.26.
[29] H. Schägger, Tricine–SDS-PAGE, Nat. Protoc. 1 (2006) 16–22, https://doi.org/10.
1038/nprot.2006.4.
[30] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem.
72 (1976) 248–254, https://doi.org/10.1016/0003-2697(76)90527-3.
[31] N. Petrovic, C. Grove, P.E. Langton, N.L. Misso, P.J. Thompson, A simple assay for a
human serum phospholipase A2 that is associated with high-density lipoproteins, J.
Lipid Res. 42 (2001) 1706–1713 (accessed June 27, 2019), http://www.ncbi.nlm.
nih.gov/pubmed/11590228.
[32] D.P. Marchi-Salvador, L.C. Corrêa, A.J. Magro, C.Z. Oliveira, A.M. Soares,
M.R.M. Fontes, Insights into the role of oligomeric state on the biological activities
of crotoxin: Crystal structure of a tetrameric phospholipase A2 formed by two
isoforms of crotoxin B from Crotalus durissus terrificus venom, Proteins Struct.
Funct. Bioinforma. 72 (2008) 883–891, https://doi.org/10.1002/prot.21980.
[33] G. Faure, H. Xu, F.A. Saul, Crystal Structure of Crotoxin Reveals Key Residues
Involved in the Stability and Toxicity of This Potent Heterodimeric β-Neurotoxin, J.
Mol. Biol. 412 (2011) 176–191, https://doi.org/10.1016/j.jmb.2011.07.027.
[34] C. Beeton, Targets and Therapeutic Properties, Handb. Biol. Act. Pept. (2013)
473–482, https://doi.org/10.1016/B978-0-12-385095-9.00064-6.
[35] I. Kerkis, M.A.F. Hayashi, A.R.B. Prieto da Silva, A. Pereira, P.L. De Sá Júnior,
A.J. Zaharenko, G. Rádis-Baptista, A. Kerkis, T. Yamane, State of the art in the
studies on crotamine, a cell penetrating peptide from South American rattlesnake,
Biomed. Res. Int. 2014 (2014) 675985, https://doi.org/10.1155/2014/675985.
[36] R.M.M. Dos Santos, L.C. Oliveira, M.I. Estevão-Costa, M.E. De Lima, M.M. Santoro,
C.L. Fortes-Dias, Inhibition of crotoxin binding to synaptosomes by a receptor-like
protein from Crotalus durissus terrificus (the South American rattlesnake), Biochim.
Biophys. Acta - Biomembr. 1717 (2005) 27–33, https://doi.org/10.1016/j.
bbamem.2005.06.014.
[37] G. Faure, D. Porowinska, F. Saul, Crotoxin from Crotalus durissus terrificus and
Crotoxin-Related Proteins: Structure and Function Relationship, in: 2017, pp. 3–20.
https://doi.org/10.1007/978-94-007-6452-1_7.
[38] L. Alves de Oliveira, R. Seabra Ferreira Jr, B. Barraviera, F. Capel Tavares de
Carvalho, L. Curtolo de Barros, L. Delazari dos Santos, D. Carvalho Pimenta,
Crotalus durissus terrificus crotapotin naturally displays preferred positions for
amino acid substitutions, (n.d.). https://doi.org/10.1186/s40409-017-0136-5.
[39] M. Waite, Chapter 9 Phospholipases, New Compr. Biochem. 20 (1991) 269–295,
https://doi.org/10.1016/S0167-7306(08)60337-3.
[40] A. Lourenço, C.F. Zorzella Creste, L. Curtolode Barros, L. Delazari dos Santos,
D.C. Pimenta, B. Barraviera, R.S. Ferreira, Individual venom profiling of Crotalus
durissus terrificus specimens from a geographically limited region: Crotamine as-
sessment and captivity evaluation on the biological activities, Toxicon. 69 (2013)
75–81, https://doi.org/10.1016/J.TOXICON.2013.01.006.