1. Biochemical Engineering Journal 53 (2011) 165–171
Contents lists available at ScienceDirect
Biochemical Engineering Journal
journal homepage: www.elsevier.com/locate/bej
Partition of lectin from Canavalia grandiflora Benth in aqueous two-phase
systems using factorial design
Camila S. Portoa,b
, Tatiana S. Portoa,b
, Kyria S. Nascimentoc
, Edson H. Teixeirad
,
Benildo S. Cavadac
, José L. Lima-Filhob
, Ana L.F. Portoa,b,∗
a
Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco, Recife, PE, Brazil
b
Laboratory of Immunopathology Keizo Asami (LIKA), Federal University of Pernambuco, Recife, PE, Brazil
c
Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, CE, Brazil
d
Faculty of Medicine, Campi of Sobral, Federal University of Ceará, Ceará, Brazil
a r t i c l e i n f o
Article history:
Received 10 March 2010
Received in revised form
29 September 2010
Accepted 7 October 2010
Keywords:
ATPS
Lectin
Canavalia grandiflora
Partition
Factorial design
PEG/citrate
a b s t r a c t
This work explores the possibility of using a polyethylene glycol (PEG)/sodium citrate aqueous two-
phase system (ATPS) as a first step in a process for the purification of lectin from Canavalia grandiflora
seed. Purification of lectins is a limiting step for its uses in the area of biotechnology. Extraction by an
aqueous two-phase system is a powerful technique for separation, concentration, and purification of
biomolecules and pharmaceutical products. Four factors (PEG’s molar mass, PEG’s concentration, pH,
citrate concentration) affecting the lectin ConGF partitioning were studied. A two-level factorial design
(24
) was carried out. Lectin ConGF preferentially partitioned to the top (polyethylene glycol) phase. The
statistical analysis showed that the citrate concentration chloride significantly affects the KL (partitioning
coefficient for protein) value for lectin partitioning. An ATPS composed of 20% (w/w) PEG 400 and 20%
(w/w) citrate, at pH 6, allowed the recovery of lectin ConGF with an 8.67 partition coefficient and 104%
yield. Consequently, the system proved to be efficient and can be used as a first step in lectin purification
from crude extract of ConGF seeds.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Lectins are carbohydrate binding proteins of non-immune ori-
gin found in all types of living organisms. They play an important
role in biological recognition phenomena, as they decipher the gly-
cocodes in the structure of glycans attached to soluble and integral
membrane glycoconjugates [1–3]. Leguminosae lectins constitute
a family proteins with related amino acid sequences that may differ
from each other strongly in their carbohydrate-binding specificity
[4]. The legume lectins from the subtribe Diocleinae, often referred
to as concanavalin A-like lectins, are a typical example of highly
similar proteins that show distinct biological activities [5].
The legume lectins from the subtribe Diocleinae, often referred
to as concanavalin A (Con A)-like lectins, are a typical example of
highly similar proteins that have distinct biological activities. Many
structural studies involving Diocleinae lectins have been performed
over the years, revealing some of the structural bases for this
∗ Corresponding author at: Department of Morphology and Animal Physiology,
Federal Rural University of Pernambuco, Rua Dom Manoel de Medeiros SN, Dois
Irmãos, 52171900 Recife, PE, Brazil. Tel.: +55 8133206345.
E-mail address: analuporto@yahoo.com.br (A.L.F. Porto).
extraordinary behavior [5–7]. Thus, the pH-dependent oligomer-
ization that some of these lectins show and the relative position of
the carbohydrate-binding site are factors that have been reported to
contribute to these differences in the activities of Diocleinae lectins
[5,8].
Several biological activities of plant lectins have been described
by many authors [9–13]. For instance, Concanavalin A (ConA) from
Jackbean (Canavalia ensiformis) have the ability to enhance protein-
specific immune responses by acting as lymphocyte mitogens, and
can also act as a relaxant on vascular smooth muscles [14,15].
Furthermore, lectins participate as adhesion molecules during anti-
inflammatory responses. Of special interest is the fact that lectin
from Canavalia grandiflora (ConGF) acts as an analgesic, and is also
a significant modulator of inflammatory events that occur on the
surface of leukocytes and vascular endothelium [16].
The purification techniques for the production of lectin are
generally based on affinity chromatography [17]. However, these
techniques are usually expensive and difficult to scale up. In this
regard, partitioning in aqueous two-phase systems (ATPS) provides
a powerful method for separating and purifying mixtures of pro-
teins [18,19]. Aqueous two-phase systems should be more efficient
mainly in large scale production of recombinant lectins because the
affinity chromatographic supports are very expensive.
1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2010.10.006

2. 166 C.S. Porto et al. / Biochemical Engineering Journal 53 (2011) 165–171
ATPSs are formed when combinations of hydrophilic solutes
(polymers or polymer and salts) display incompatibility in aqueous
solution above critical concentrations [20]. Extraction by aqueous
two-phase systems has become an important emerging technique
for separation, concentration and purification of proteins, enzymes
and pharmaceutical products [21].
Production costs of biological products (50–90%) are determined
by the purification strategy. The downstream processing of bio-
logical material requires separation and purification techniques
leading to a high degree of purification and recovery and to low
operating costs. For this reason, the development of techniques and
methods for the separation and purification of proteins has been
an important prerequisite for many of the advances made in the
biotechnology industry [22].
Aqueous two-phase systems have been successfully used for
separation and purification of several proteins and show signifi-
cant advantages over traditional methods [23], such as the high
water content of phases, signifying high biocompatibility, thereby
minimizing biomolecule degradation, low interfacial tension, low-
cost and easily scaled-up [22–24]. Thus, this method constitutes an
interesting alternative to the traditional downstream processing
of lectins since clarification, concentration and purification can be
combined in only one operation using a non-toxic phase environ-
ment [25,26].
Well-studied two-phase systems include polyethyleneglycol
(PEG)/dextran and PEG/sal, where each phase generally contains
80–90% (w/w) water, and provides a gentle environment for pro-
teins, nucleic acids, viruses and other biological molecules [25]. For
industrial purposes, polymer/potassium phosphate systems are the
most commonly used, but the high salt concentration required rep-
resents a waste disposal problem which leads to environmental
concerns [22]. Previous studies have demonstrated that replac-
ing the inorganic salts by other biodegradable and non-toxic ones
such as citrates could be considered a good alternative, because cit-
rates can be discharged into biological wastewater treatment plants
[22,26–28].
Few studies have been published with lectins applied to
liquid–liquid extraction [29–31]. The use of purified lectins in ATPS
is justified because it allows the study of biomolecule partition,
allowing the use of the same conditions in the recovery of lectins
from the crude extract. Therefore, the aim of this work was to study
the partition of the ConGF lectin in an aqueous two-phase system
PEG/citrate using 24 full factorial designs.
2. Materials and methods
2.1. Chemicals
All chemicals used in this work were of analytical grade and
were obtained from Merck (Darmstadt, Germany) and Sigma (St.
Louis, MO).
2.2. Lectin from C. grandiflora
A d-glucose/d-mannose-specific lectin from seeds of C. grandi-
flora (ConGF) was purified by affinity chromatography on Sephadex
G-50 as previously described by Ceccatto et al. [17]. By sodium
dodecyl sulfate polyacrylamide gel electrophoresis, ConGF yielded
three protein bands with apparent molecular masses of 29–30 kDa
(alpha chain), 16–18 kDa (beta fragment), and 12–13 kDa (gamma
fragment), like other related lectins from the genus Canavalia (Legu-
minosae), and is endotoxin free [16].
Table 1
Variable levels of the 24
experimental design used for the ConGF lectin partition by
ATPS.
Variables Low (−1) Central (0) High (+1)
PEG molar mass (g/mol) 400 3350 8000
PEG concentration (%, w/w) 20.0 22.0 24.0
Citrate concentration (%, w/w) 15.0 17.5 20.0
pH 6.0 7.0 8.0
2.3. Protein assays
Protein content was determined by the ultraviolet spectropho-
tometric method with absorbance measurements taken at 260
and 280 nm according to Warburg and Christian [32]. In order to
avoid interference from PEG and citrate, the samples were analyzed
against blanks containing the same phase composition voided of
protein.
2.4. Determination of the hemagglutinating activity
The determination of the hemagglutinating activity (HA) in the
aqueous phase was performed in microtiter plates [33]. Lectin
preparations (50 ␮l) were two-fold serial diluted with 0.15 M NaCl
before addition of 50 ␮l suspension of rabbit erythrocytes (2%)
treated with glutaraldehyde 2.5% (v/v), the mixture incubated
at 25 ◦C for 30 min. The hemagglutination titer (HU, mL−1) was
recorded as the reciprocal of the highest dilution giving visible
agglutination. This concentration was denoted as containing one
hemagglutinating unit (HU) per mL [17].
2.5. Preparation of aqueous two-phase systems
A sodium citrate concentrated solution (30% (w/w)) was pre-
pared by mixing appropriate amounts of solutions of tri-sodium
citrate dihydrate and citric acid monohydrate at different pH values
(6.0, 7.0, 8.0), at 25 ± 1 ◦C. The required amount of the previous solu-
tion was mixed, in 15 mL graduated tubes with conical tips, with
50% (w/w) PEG solution and lectin solution (0.5 mg/mL) represent-
ing 20% (w/w) of total mass. Water was added to a final amount of
5 g. After vortex shaking for 1.0 min, the two phases were separated
by settling for 40 min. Phase were then measured; top and bottom
phases were separately withdrawn with pipettes and assayed for
protein concentration and hemagglutinating activity.
2.6. Experimental design and statistical analysis
The influence of variables, namely PEG molar mass (MPEG), PEG
concentration (CPEG), citrate concentration (CC) and pH, on the two
selected responses, namely, activity yield (Y) and partition coeffi-
cient (K), was evaluated from the results obtained with 24 factorial
designs plus a central point, which was run in quadruplicate to
allow estimation of pure experimental error [34]. Levels of vari-
ables assessed in the 24 factorial ATPS design used in this study
were chosen according to Porto et al. [26], and are presented in
Table 1. All statistical and graphical analyses were carried out with
the Statistica 8.0 program [35].
2.7. Determination of partition coefficient and yield
The activity partition coefficient of ConGF lectin was calculated
as the ratio of the hemagglutinating activity in the top phase to that
of the bottom phase.
KHA =
HAt
HAb
(1)

3. C.S. Porto et al. / Biochemical Engineering Journal 53 (2011) 165–171 167
Table 2
Conditions and results of the 24
-experimental design selected for ConGF lectin partitioning by PEG/citrate ATPS.
Runs MPEG CPEG CC pH KL KHA YL (%) YHA (%)
1 400 20 15 6 – – – –
2 8000 20 15 6 2.25 1.00 68.7 262.50
3 400 24 15 6 – – – –
4 8000 24 15 6 2.30 1.00 100.4 300.00
5 400 20 20 6 8.67 1.00 104.0 137.50
6 8000 20 20 6 4.60 1.00 83.6 125.00
7 400 24 20 6 2.86 1.00 89.1 153.13
8 8000 24 20 6 4.71 2.00 134.0 300.00
9 400 20 15 8 – – – –
10 8000 20 15 8 0.92 0.25 45.8 262.50
11 400 24 15 8 – – – –
12 8000 24 15 8 1.73 2.00 86.4 312.50
13 400 20 20 8 6.75 4.00 103.1 525.00
14 8000 20 20 8 2.09 4.00 71.1 425.00
15 400 24 20 8 3.71 2.00 113.5 300.00
16 8000 24 20 8 3.71 8.00 118.2 625.00
17 (C) 3350 22 17.5 7 0.34 2.00 8.7 275.00
18 (C) 3350 22 17.5 7 0.71 1.00 27.3 137.50
19 (C) 3350 22 17.5 7 0.30 1.00 16.0 137.50
20 (C) 3350 22 17.5 7 1.56 2.00 42.8 137.50
MPEG = PEG molar mass; CPEG = PEG concentration; CC = citrate concentration; KL = partition coefficient of ConGF lectin; KHA = partition coefficient of hemagglutinating activity;
YL = ConGF lectin yield; YHA = hemagglutinating activity yield; (C) = central points; Bold values represent the best conditions for Lectin ConGF partition.
where HA is the lectin hemagglutinating activity (HU, mL−1). The
subscripts “t” and “b” represent the top and bottom phases, respec-
tively. Similarly partitioning coefficient for protein (KL) is given
by:
KL =
Ct
Cb
(2)
where Ct and Cb are the protein concentrations in top and bottom
phases, respectively, expressed in mg/mL.
The activity yield (YHA) was determined as the ratio of total activ-
ity in the top phase to that in initial lectin solution and expressed
as percentage:
YHA =
HAt · Vt
HAi · Vi
× 100 (3)
where HAt and HAi, are the hemagglutinating activities in the top
phase and initial lectin solution, respectively. Similarly Yield for
protein (YL) is given by:
YL =
Ct · Vt
Ci · Vi
× 100 (4)
where Vt and Vi are the volumes of the top phase and the initial
lectin solution, respectively.
3. Results and discussion
The strategy used to attain the goal of this work was to explore
the influence of PEG molar mass and concentration, citrate con-
centration and pH on ConGF lectin partitioning and yield in a
PEG/citrate ATPS. Results are presented in Table 1. The tests that
did not form two aqueous phases (Table 2, test 1, 3, 9 and 11) are
below the binodal curve obtained with PEG 400 g/mol by Porto et al.
[26]. Concentrations below the binodal curve for PEG 400 were
selected for this study because the concentrations for the forma-
Fig. 1. Pareto chart for the standardized effects of the variables: (1) PEG molar mass – MPEG, (2) PEG concentration – CPEG, (3) citrate concentration – CC and (4) pH on partition
coefficient of ConGF lectin. The extension of bars across the vertical dotted line (p = 0.05) represents the dimensions of significance.

4. 168 C.S. Porto et al. / Biochemical Engineering Journal 53 (2011) 165–171
Fig. 2. Cubic plot of the effects on the partition coefficient (KL) obtained according to the design in Table 1.
tion of phases in this system are very high, which would increase
the viscosity of the PEG 8000, affecting the extraction of the lectin.
Therefore, systems with PEG 400 did not form two phases, but no
problems were observed with PEG 8000 at the same concentration.
The partition coefficient (K) of ConGF lectin was higher than
1, indicating that lectin ConGF preferentially partitioned to the
top phase. As with the lectin partition coefficient (KL), hemagglu-
tinating activity (KHA) was also equal to or higher than 1. Zhang
et al. [30] studied the partition of Ricin (lectin) in PEG/sulfate and
PEG/phosphate systems and observed that the lectin partitioned to
the PEG phase (partition coefficient greater than 1) in both systems.
Although the efficiency of partitioning depend on a number of
factors such as hydrophobic properties, addition of salts, electrical
potential between the phases, molecular size and molecules con-
formation, the hydrophobic characteristics are considered to be the
dominant influencing factor [36]. Specifically, in PEG-salt systems
Fig. 3. Pareto chart for the standardized effects of the variables: (1) PEG molar mass – MPEG, (2) PEG concentration – CPEG, (3) citrate concentration – CC and (4) pH on yield
of ConGF lectin (YL). The extension of bars across the vertical dotted line (p = 0.05) represents the dimensions of significance.

5. C.S. Porto et al. / Biochemical Engineering Journal 53 (2011) 165–171 169
partitioning depends on the “volume exclusion effect” in the poly-
mer rich phase (top) and the “salting out effect” in the salt rich
phase (bottom). The volume occupied by the polymer increases
with polymer concentration and molar mass, leading to a decrease
in the amount of space available for the molecules in the top
phase. This phenomenon known as the “volume exclusion effect”
causes biomolecules to have the tendency to partition to the bottom
phase. In contrast, the solubility of biomolecules decreases with
the increase in the salt concentration found in the phase rich in salt
(bottom), which leads to the increased partitioning of biomolecules
to the top phase known as the “salting out effect” [37–39].
Statistical analyses of the responses revealed that some data
were statistically significant at the 95% confidence level. The Pareto
chart showing the partition coefficient of ConGF (Fig. 1) shows that
the citrate concentration has the greatest positive effect, which
means that the highest level of CC provided the highest value of KL
(8.67). The highest partition coefficients obtained for ConGF is com-
parable to the partition coefficient of Ricin (lectin) in PEG/sodium
sulfate (4.5–14.5) [30]. Salts can change the electrostatic charge
of ATPS and influence the distribution of charged amino acids or
proteins [40]. With an increase in citrate concentration, negatively
charged proteins prefer the PEG rich phase because of repulsion
force caused by salt anions [41]. This phenomenon was observed
by Porto et al. [38], when partitioning a protease from Clostridium
perfringens in PEG/citrate. They observed that protease transferred
to the top phase as citrate concentration increased.
At higher salt concentrations, the ions decrease protein sol-
ubility (salting-out effect) by increasing hydrophobic interaction
and aggregation, and the hydration effect of the salt molecule sur-
rounding the protein. Therefore, most proteins strongly partition
to the phase with lower salt concentration, thereby increasing the
interaction between the protein and the PEG molecules and ulti-
mately improving extraction of proteins to the PEG-rich phase [36].
Significant interactions between PEG molar mass (MPEG), PEG con-
centration (CPEG), and citrate concentration (CC), are presented in
the cubic plot (Fig. 2) with the observed responses are displayed
against combinations of varying levels of the three variables. The
highest KL values (8.67 and 6.75) are located on the top face of the
cube, and these correspond to 20% citrate, 20% PEG, and PEG molar
mass 400 g/mol.
For both parameters KL and KHA, the variable that most influ-
enced the partition coefficient was citrate concentration with
positive effect, such that the highest citrate concentration in the
bottom phase favored the displacement of the ConGF lectin to the
top phase. For the KHA, the pH and PEG molar mass also had sig-
nificant positive effects, indicating that higher pH and molar mass
values increased the KHA.
The pH presented a statistically significant effect for KHA. It may
have influenced the partition of the ConGF to top phase because, the
lectin has isoeletric point in the acidic region [42], and therefore has
a negatively charged surface under the pH values used in this study
(6.0, 7.0 and 8.0), thus enhancing its move to the top positive phase,
through electrostatic interactions between the protein and PEG
molecules. Several authors have reported that negatively charged
proteins partition to the top phase (PEG) and positively charged
proteins to the bottom phase [19,41,43,44]. The pH effect can be
explained by isoelectric point and the pH-dependent oligomeriza-
tion of legume lectins. It has been reported that Diocleinae lectins
are structurally active in a state of dimer–tetramer equilibrium in
the range of pH around 4.5–8.5 with the content of tetrameric form
increasing with the pH [5,45].
According to Table 2, it appears that ConGF lectin was recov-
ered mainly in the PEG-rich phase as indicated by both protein (YL)
and hemagglutinating activity (YHA). Statistical analysis performed
showed that citrate concentration and PEG molar mass are the two
main factors that significantly affected yield (Fig. 3), thereby thus
Fig. 4. Simultaneous effects of citrate concentration – CC (%, w/w) and PEG molar
mass – MPEG (g/mol) on the yield – YL (%) of lectin from C. grandiflora by PEG/citrate
ATPS. Experiments performed according to the 24
-factorial design in Table 1.
corroborating the fact that CC and MPEG provided the best values of
YL. Similarly, a positive interaction effect was observed between CC
and MPEG, indicating a synergism between the two variables. Thus,
higher YL values will be obtained if citrate concentration and PEG
molar mass are raised simultaneously. The highest yield values of
lectin from C. grandiflora are indeed observed at the 8 and 16 exper-
imental runs, as shown in Fig. 4. Malpiedi et al. [22] found that the
trypsinogen yield increased when high polymer molar mass were
used in the ATPS. However, there is a relationship between YL and
YHA, both were positively influenced by PEG molar mass and cit-
rate concentration, i.e. the best conditions for YL and YHA had PEG
8000 g/mol and 20% of citrate.
In some systems, the lectin yield (YL) had values above 100%, be
due to interference of the system components in the protein anal-
ysis. Also, high values of hemagglutinating activity yield (YHA) can
possibly be explained by an activation lectin site in the presence of
PEG. There are several studies that describe the structure of legume
lectins and the high homology between them [44]. An important
feature is the existence of hydrophobic sites and hydrophobic cav-
ities, which possibly interacted with the PEG, increasing the values
of hemagglutinating activity.
Hydrophobic cavities probably have an extreme importance
for the lectin partition for the more hydrophobic phase (PEG rich
phase). In the presence of PEG, C. ensiformis (ConA) has been
reported to have a compact structure that is characterized by exter-
nal hydrophobic regions which facilitates interaction with the PEG
in the solution. As PEG is hydrophobic in nature, it may interact
favorably with the hydrophobic side chains exposed upon unfold-
ing. So, most probably, the PEGs perturb the structure of the lectin
surface, where precise topology is required for their biological
activities [45]. In this study, this behavior was confirmed because
the ConGF lectin showed a high affinity for the PEG-rich phase.
In order to determine the optimum partition coefficient of
ConGF lectin in ATPS, the responses (KL and YL) were compared
(Table 2). Accordingly, conditions were selected to meet the fol-
lowing two requirements: (i) to have K > 5, and (ii) to have Y
around 100%. The runs 5 (KL = 8.67 and YL = 104%) and 13 (KL = 6.75
and YL = 103%) were selected as the best ones for ConGF lectin
extraction. Both runs have PEG molar mass 400 g/mol, PEG concen-

6. 170 C.S. Porto et al. / Biochemical Engineering Journal 53 (2011) 165–171
tration of 20%, citrate concentration of 20%, differing only on the pH
value.
4. Conclusion
The ConGF lectin preferentially partitioned to the PEG phase
and the ATPS were strongly influenced by citrate concentration
and PEG molar mass due to salting out and the volume exclusion
effects, respectively. The factorial design proved that PEG 400 g/mol
(20%) and citrate concentration of 20% showed the best results for
ConGF lectin partitioning in ATPS PEG/citrate. These results open
the possibility of using ATPS to purify Leguminosae lectins from
crude extract.
Acknowledgments
The authors wish to acknowledge the financial support and
scholarship of CAPES (Coordenac¸ ão de Aperfeic¸ oamento de Pessoal
de Nível Superior, Brasilia, Brazil) and CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico, Brasilia, Brazil).
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