520 Protein & Peptide Letters, 2012, 19, 520-529

Antimicrobial Peptides from Adenanthera pavonina L. Seeds: Characteri-

zation and Antifungal Activity

Julia Ribeiro Soares1,¶, André de Oliveira Carvalho1,¶, Izabela Silva dos Santos1, Olga Lima Tavares
Machado2, Viviane V. Nascimento1, Ilka Maria Vasconcelos3, André Teixeira da Silva Ferreira4,
Jonas Enrique de Aguilar Perales4 and Valdirene Moreira Gomes1,*

1
Laboratório de Fisiologia e Bioquímica de Microrganismos, Universidade Estadual do Norte Fluminense, Campos dos
Goytacazes, RJ, Brazil; 2Laboratório de Química e Função de Proteínas e Peptídeos, Universidade Estadual do Norte
Fluminense, Campos dos Goytacazes, RJ, Brazil; 3Laboratório de Toxinas Vegetais, Universidade Federal do Ceará,
CE, Brazil; 4Oswaldo Cruz, Rio de Janeiro, RJ, Brazil

Abstract: In this study, the antifungal activity of peptides extracted from Adenanthera pavonina seeds was assessed. Pep-
tides were extracted and fractionated by DEAE-Sepharose chromatography. The non-retained D1 fraction efficiently in-
hibited the growth of the pathogenic fungi. This fraction was later further fractionated by reversed-phase chromatography,
resulting in 23 sub-fractions. All separation processes were monitored by tricine-SDS-PAGE. Fractions H11 and H22
strongly inhibited the growth of Saccharomyces cerevisiae and Candida albicans. Fraction H11 caused 100% death in S.
cerevisiae in an antimicrobial assay. The complete amino acid sequence of the peptide in fraction P2 was determined, re-
vealing homology to plant defensins, which was named ApDef1. Peptides from fraction H22 were also sequenced.
Keywords: Adenanthera pavonina, Candida albicans, defensin, fungicide activity, membrane permeabilization, Saccharomy-
ces cerevisiae.

INTRODUCTION [13], snakins [14] and cyclotides [15]. These antimicrobial

Innate immunity is important for the survival of multicel-
lular organisms under threat by invading microorganisms. A
key component of innate host defense is the production of
antimicrobial peptides (AMPs) [1]. Indeed, virtually every
form of life, from bacteria on, produces these self-defense
molecules [1]. AMPs are low molecular weight amphiphilic
peptides that contain a positive net charge at physiological
pH and are endowed with antimicrobial activity [2]. AMPs
may be constitutively expressed or inducibly produced upon
sensing an invader, and their antimicrobial effect is primarily
mediated through interaction with biological membranes. In
addition to this primary target, intracellular targets have al-
ready been described for some AMPs, including binding to
chaperones [3], interfering with macromolecular synthesis
[4] and disrupting the cell cycle by interacting with cyclin F
[5]. For the majority of AMPs, the mechanism of action
leading to microbial growth inhibition remains unknown.
In plants, several families of AMPs have been described
and characterized; most of these families are rich in disul-
fide-forming Cys residues. These disulfide bonds endow
AMPs with great physicochemical stability [6,7]. Plant-
derived AMPs include lipid transfer proteins [8, 9], thionins
[10], plant defensins [11], hevein-like peptides, knottin-like
peptides, glycine-rich peptides [12], MBP-1 homologs
*Address correspondence to this author at the Laboratório de Fisiologia e
Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia,
Universidade Estadual do Norte Fluminense, Campos dos Goytacazes,
28013-602, RJ, Brazil; Tel: +55 22 2739-7033; Fax: +552227397178;
E-mail: valmg@uenf.br
¶
These authors contributed equally to this work.
substances are particularly abundant in seeds [16], where
they are constitutively expressed during normal development
and participate in protection against microorganisms in soil
[17].
In recent decades, AMPs have been considered a promis-
ing source of new pharmaceuticals [18], particularly given
increased bacterial resistance to conventional antibiotics
[19], inadequate investment in the development of new anti-
biotics [20] and increased cases of invasive mycoses in im-
munocompromised patients, primarily those caused by Can-
dida spp. [21]. In particular, plant defensins have been sug-
gested as a possible new source of therapeutics to treat inva-
sive mycoses [22].
Previous studies demonstrated that A. pavonina seeds
contain a chitinase with robust thermal stability [23, 24]. The
present report contains an analysis of the AMPs present in A.
pavonina seeds. The purification of a peptide with homology
to the plant defensin family that exhibits potent inhibitory
activity against S. cerevisiae is reported.
MATERIAL AND METHODS
Plant Materials
Seeds from Adenanthera pavonina (L.) were collected in
fields of Campos dos Goytacazes province, Rio de Janeiro,
Brazil.
Fungi
Saccharomyces cerevisiae (1038), Candida albicans
(CE022) and Candida tropicalis (CE017) were obtained

-5/12 $58.00+.00 © 2012 Bentham Science Publishers

Antimicrobial Peptides from Adenanthera pavonina
from the Departamento de Biologia, Universidade Federal do
Ceará, Fortaleza, Brazil. Fusarium oxysporum (FBM 002)
was kindly provided by CNPAF/EMBRAPA, Goiania,
Goiás, Brazil. Fungi were maintained on Sabouraud agar
(1% peptone, 2% glucose and 1.7% agar-agar).
Extraction and Purification of Antimicrobial Peptides
Purification of peptides from A. pavonina seeds was per-
formed as described previously with modifications [25]. Pep-
tides from seed flour (50 g) were extracted for 3 h (4 ºC)
with 500 mL of extraction buffer (10 mM Na2HPO4, 15 mM
NaH2PO4, 100 mM KCl and 1.5% EDTA, pH 5.4). The pre-
cipitate formed between 0 and 90% relative ammonium sul-
fate saturation, after centrifugation (15000 x g, 30 min), was
redissolved in distilled water and heated at 80 ºC for 15 min.
The resulting suspension was clarified by centrifugation
(10000 x g, 10 min) and the supernatant was extensively
dialyzed against distilled water. The dialyzed solution was
recovered by freeze drying (fraction F/0-90) and was further
used for antimicrobial peptides purification.
Separation of proteins from fraction F/0-90 was carried
out using a DEAE-Sepharose column (3 x 7 cm, GE
HealthCare) equilibrated with 100 mM Tris-HCl (pH 8.0).
The column was eluted at a flow rate of 90 mL/h, first with
equilibrium buffer to obtain fraction D1 and then with equi-
librium buffer containing 1 M NaCl to obtain bound proteins
(fraction D2). Protein elution was monitored by measurement
of absorbance at 280 nm (Spectrophotometer LGS53, BEL
Photonics).
Fraction D1 was recovered by freeze drying (FreeZone
4.5, Labconco), redissolved in 0.1% (v/v) trifluoroacetic acid
(TFA, Fluka) and loaded onto an HPLC (Prominence, Shi-
madzu) C18 reversed-phase column (250 x 4.6 mm, Shima-
dzu) attached to a C8 pre-column (20 x 4.6 mm, Pelliguard,
Sigma-Aldrich). The solvent flow rate was 0.5 mL/min, and
the following solvent gradient was used: 100% solvent A
(0.1% TFA in water) for 7 min, 0 to 42% solvent B (100% 2-
propanol containing 0.1% TFA) for 64 min, 50% solvent B
for 2 min and 0% solvent B for 10 min. Elution was moni-
tored by online measurement of absorbance at 220 and 280
nm.
Fraction H11 was diluted in 0.1% (v/v) trifluoroacetic
acid and loaded onto an HPLC C2C18 reversed-phase col-
umn (100 x 4.6 mm, μRPC ST 4.6/100, GE HealthCare). The
solvent flow rate was 0.5 mL/min, and the following solvent
gradient was used: 100% solvent A for 5 min, 0 to 17% sol-
vent B for 10 min, 22% solvent B for 10 min and 0% solvent
B for 10 min. Elution was monitored by online measurement
of absorbance at 220 and 280 nm.
Protein content was determined as described by Bradford
[26] with albumin from chicken egg white (Sigma) used as
the protein standard.
Electrophoresis
Tricine-sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (tricine-SDS-PAGE) was performed according to
the method of Schägger and von Jagow [27].
Protein & Peptide Letters, 2012, Vol. 19, No. 5 521
Amino Acid Sequence Analysis
Amino acid sequence analysis was performed on peptides
isolated from fractions H11 and H22. These fractions were
treated with 5 mM
-mercaptoethanol (Sigma) and 5%
vinylpyridine (Sigma) and were subsequently heated for 30
min at 37 ºC and 15 min at 60 ºC. After treatment, the sam-
ples were separated by tricine-SDS-PAGE, transferred to
polyvinylidene difluoride (PVDF, Millipore) membranes and
stained with Ponceau-S (0.1%, GE HealthCare) [28]. The
band corresponding to approximately 7 kDa from fraction
H11 and the bands at approximately 3 and 6 kDa from frac-
tion H22 were excised from the corresponding membrane;
each was briefly washed three times in water, and the mem-
brane was air-dried. N-terminal amino acid sequences of
peptides blotted onto PVDF were determined by Edman deg-
radation carried out in a Shimadzu PSQ-23A protein se-
quencer (Shimadzu). Phenylthiohydantoin (PTH) –derivati-
zed amino acids were detected at 269 nm after separation on
a reversed-phase C18 column (250 x 4.6 mm) under isocratic
conditions according to the manufacturer's instructions.
Searches for sequence homology were performed using
BLASTp [29], and selected sequences were aligned with
ClustalW [30].
The complete sequence of the lower molecular weight
peptide of fraction H11, referred to as P2, was determined by
proteolytic digestion followed by N-terminal sequencing.
The purified peptide was dissolved in 100 mM Tris-HCl, pH
7.8, the endoproteinase Glu-C (Sigma) was added at a 1:50
ratio (μg:μg) (H11 endoproteinase:sample), and the solution
was incubated at 37 ºC for 16 h. After this treatment, the Cys
residues in the peptide fragments were reduced and purified
by reversed-phase HPLC as described previously using a
C18 column attached to a C8 guard column. The solvent
flow rate was 0.5 mL/min, and the following solvent gradient
was used: 100% solvent A for 10 min, 0 to 60% solvent B
for 130 min, 60 to 100% solvent B for 5 min and 0% solvent
B for 15 min. Elution was monitored by online measurement
of absorbance at 220 and 280 nm. The eluted sample was
dried in a Speed Vac Plus (SC110A, Savant). Two main
peaks were obtained after cleavage. The corresponding frac-
tions were subjected to N-terminal amino acid sequence
analysis, and sequence homology searches were performed
as described above. In addition, the purified peptide, in both
native form and reduced, alkylated form, was subjected to
mass spectrometry analysis. Peptide was cocrystalized with
an excess of the matrix alpha cyano-4-hydroxycinnamic acid
and further analyzed by matrix-assisted laser desorption ioni-
sation time-of-flight mass spectrometry (MALDI-TOF MS).
The instrument used was an AB SCIEX TOF/TOF™ 5800
System spectrometer (AB SCIEX) in the linear mode.
Effect of Fraction D1, H11 and H22 on Fungi Growth
Fungus and yeast cultures were prepared by transferring
an inoculum of each to Petri dishes containing Sabouraud
agar and allowing them to grow at 30 ºC for 48 h. After this
period, the cells were transferred to sterile Sabouraud broth
(1 mL). Yeast cells were counted in a Neubauer chamber for
further calculation of appropriate dilutions. To monitor the
effect of AMPs from A. pavonia seeds on the growth of
yeasts, 104 cells/mL (in Sabouraud broth) were incubated in
522 Protein & Peptide Letters, 2012, Vol. 19, No. 5
the presence of 25, 50 and 100 μg/mL peptide at 28 ºC in
200 μL total volume in 96 well microplates. Culture medium
was used as the blank for the spectrophotometer. Optical
readings were taken at 670 nm at the zero time point and
every 6 h for the following 60 h for fungi and 36 h for yeasts
[31]. Optical densities were plotted as a function of absor-
bances.
Effect of Fractions H11 and H22 on Yeast Cell Viability
After yeast cultures were grown as described above for
36 h, cells in the untreated control wells were quantified in a
Neubauer chamber as a control dilution. The untreated con-
trol and test samples (treated with 80 μg/mL of each frac-
tion) were diluted in Sabouraud broth as appropriate and
plated with a Drigalski loop on Petri dishes containing
Sabouraud agar. Cells were evenly distributed on the plate
and were incubated at 30 ºC for 48 h to allow colony devel-
opment. After this period, the colonies were counted. Data
were analyzed with respect to the untreated control sample,
which was considered to be 100% viable.
Plasma Membrane Permeabilization
Plasma membrane permeabilization was measured by
SYTOX® Green uptake as described previously by Thevis-
sen et al. [32] with modifications. After fungal and yeast
cultures were grown for 36 h as described above, 100 μL
aliquots of S. cerevisiae, C. albicans and C. tropicalis sus-
pensions that were grown in the presence of the AMP-
containing fractions from A. pavonina were further incubated
with 0.2 μM SYTOX® Green in 96 well microplates for 1.5
h at 25 ºC with periodic agitation. Cells were then observed
using a DIC microscope (Axiophoto Zeiss) equipped with a
fluorescence filter set for fluorescein detection (excitation
Figure 1. Chromatogram of the fraction F/0-90 in DEAE-Sepharose column. D1 was eluted with equilibrium buffer (Tris-HCl 0.1 M, pH
8.0), and D2 was eluted with equilibrium buffer containing 1 M NaCl. The arrow indicates the buffer change. Insert: Electrophoretic visuali-
zation of fraction F/0-90 and fractions D1 and D2 in tricine-SDS-PAGE. F/0-90, fraction obtained after 90% relative ammonium sulfate satu-
ration. Fraction D1, not retained in the DEAE-Sepharose column; fraction D2, retained in the DEAE-Sepharose column and eluted with 1 M
NaCl; M, molecular weight markers indicated in kDa.
Soares et al.
450-490 nm; emission 500 nm). An untreated negative con-
trol was included to evaluate the baseline membrane perme-
ability, and a positive control was included that contained
cells killed by heating at 100 ºC for 5 min.
RESULTS
Extraction and Partial Purification of A. pavonina An-
timicrobial Peptides
Fraction F/0-90 from A. pavovina seeds was separated
into two different fractions, D1 and D2, by anion exchange
chromatography on a DEAE-Sepharose column Fig. (1).
Tricine-SDS-PAGE analysis showed that these fractions
contained peptides with molecular weights between 6 and 10
kDa and that they contained several proteins with molecular
weights above 16 kDa (Fig. 1 insert).
Fraction D1 was further fractionated into 23 fractions by
reversed-phase chromatography (RP) on a C18 column; one
fraction contained the flow-through, and the other 22 frac-
tions contained material eluted from a propanol gradient Fig.
(2). With the exception of some peptides in fraction 22, all
peptides detected were between 2 and 17 kDa Fig. (3).
Among the 22 isopropanol gradient fractions, two criteria
were used to choose the fractions that were tested for antimi-
crobial activity: the homogeneity of the fraction and the
presence of low molecular weight peptides. Using these cri-
teria, fractions H11, H17, H18, H19, H21 and H22 were selected
for further analysis. Only fractions H11 and H22 exhibited
antimicrobial activity (results not shown for other fractions).
Characterization of the Antimicrobial Activity
The antimicrobial assay demonstrated that the fraction D1
possessed antimicrobial activity, which was expected
Antimicrobial Peptides from Adenanthera pavonina
Figure 2. C18 reversed-phase HPLC chromatogram of fraction D1. D1 was fractionated into 23 fractions using a propanol/TFA gradient
(oblique line) at a flow rate of 0.5 mL/min. Fractions were named as unbound and H1 to H22. Elution was monitored at 220 nm.
Figure 3. Electrophoretic visualization of the 22 bound fractions in
reversed-phase C18 column in tricine-SDS-PAGE. 1 to 22, bound
fractions and eluted upon propanol gradient formation. M, molecu-
lar weight markers are indicated in kDa.
Protein & Peptide Letters, 2012, Vol. 19, No. 5 523
because of the basic character of the peptides present in this
fraction. At the endpoint of the antimicrobial assay, fraction
D1 demonstrated approximately 37, 53 and 82% growth in-
hibition of the fungus F. oxysporum at 25, 50 and 100
μg/mL, respectively Fig. (4A). Fraction D1 did not inhibit
growth of S. cerevisiae Fig. (4B). At the endpoint of the as-
say, growth inhibition of C. tropicalis was 32 and 79% at the
concentrations 50 and 100 μg/mL, respectively Fig. (4C).
Inhibition of C. tropicalis growth was not observed at 25
μg/mL.
At 80 μg/mL, fraction H11 inhibited growth of the yeasts
S. cerevisiae and C. albicans by approximately 83% and
42%, respectively at the assay endpoint Fig. (5A and 5B).
Fraction H22 inhibited growth of S. cerevisiae by approxi-
mately 22% at a concentration of 80 μg/mL Fig. (6A) and
did not inhibit growth of C. albicans Fig. (6B).
To characterize the mechanism of the antimicrobial ef-
fects of the H11 and H22 fractions, we also tested the effects
of these fractions on yeast membrane permeabilization and
cell viability. The membrane permeabilization assay was
performed immediately after the growth assay for the yeasts
S. cerevisiae and C. albicans, using the nucleic acid stain
SYTOX® Green. Cells treated with fractions H11 and H22 did
not exhibit an increase in fluorescence when compared with
the untreated control, indicating that the membranes of these
cells were not permeabilized by the peptides present in the
fractions (Table 1).
After the growth assay, cells from the control were
counted, diluted and plated on Sabouraud agar. Similarly,
cells treated with the H11 fraction were diluted and plated. S.
cerevisiae cells treated with fraction H11 exhibited 100%
death Fig. (7A and B), whereas C. albicans cells exhibited
0% death Fig. (7C and D).
Purification of Peptides of H11 and Amino Acid Sequence
of P2 and H22 Fractions
To identify the peptides contained in fraction H11, the
mixture was submitted to C2C18 RP chromatography, result-
524 Protein & Peptide Letters, 2012, Vol. 19, No. 5 Soares et al.

Figure 5. Growth curves of the yeasts Saccharomyces cerevisiae

(A) and Candida albicans (B) in the presence of fraction H11. ()
control; () 80
g/mL, (n = 3).

Figure 4. Growth curves of the filamentous fungus Fusarium ox-

ysporum (A) and the yeasts Saccharomyces cerevisiae (B) and
Candida tropicalis (C) in the presence of fraction D1. () control;
() 25
g/mL; () 50
g/mL and () 100
g/mL, (n = 3).

ing in three peaks Fig. (8). Peak P2 contained a single peptide

of approximately 7 kDa, which corresponds to the ApDef1 ,
and P3 contained a single peptide of approximately 17 kDa
(Fig. (8) insert).
N-terminal amino acid sequence analysis of the lower
molecular weight peptide of fraction H11, approximately 7
kDa in size, showed a homology with the primary structure
of antimicrobial peptides from the plant defensin family (re-
sult not shown). To obtain the complete amino acid sequence
of P2, the purified sample was digested with endo Glu-C, and
the proteolytic fragments were purified. Two main peaks
were obtained after cleavage. These two peaks were sub-
jected to N-terminal amino acid sequence analysis, and pro-
tein sequence databases were queried to identify similar pep-
tides. This analysis revealed that we had determined the first
31 and the last 17 amino acids of a peptide from plant de-
fensin family. The first 31 amino acids were determined to Figure 6. Growth curves of the yeasts Saccharomyces cerevisiae
arise directly from the peptidyl N-terminus. By combining (A) and Candida albicans (B) in the presence of the fraction H22.
() control; () 80
g/mL, (n = 3).
Antimicrobial Peptides from Adenanthera pavonina
Figure 7. Cell viability assay of Saccharomyces cerevisiae (A,
control and B, test) and Candida albicans (C, control and D, test)
treated with fraction H11.
this information with the database query results and the
known substrate specificity of endo Glu-C, we were able to
determine the endo Glu-C cleavage site and the complete
peptide sequence. In addition, we obtained a mass of 5129
Da for the intact peptide and 5590 Da for the intact alkylated
peptide, consistent with the presence of eight Cys residues
(result not shown). The amino acid sequence of P2 exhibited
a 72, 41 and 42% homology with the defensins 5144 (from
Cassia fistula), PsD2 and NsD1, respectively Fig. (9A).
The 21 amino acid N-terminal sequence obtained from
the 6 kDa peptide contained in fraction H22 showed a homol-
ogy with the primary structure of the ubiquitin family. It
exhibited 95% identity with ubiquitin from Oryza sativa,
Arabidopsis thaliana and Daucus carota Fig. (9B). The 18
amino acid N-terminal sequence obtained from the 3 kDa
peptide (SNYDYDVETETAYAYT) contained in fraction
H22 revealed no sequence homology with any other known
peptide, even those isolated from other plant seeds. Further
Protein & Peptide Letters, 2012, Vol. 19, No. 5 525
analyses to characterize this peptide more fully are under-
way.
DISCUSSION
Most AMPs are characterized by low molecular weight,
amphipathicity and basic character. A purification strategy
was developed to isolate peptides with these properties from
A. pavonina seeds based on an extraction and purification
method that was previously used to isolate AMPs from seeds
of Raphanus sativus [25]. Other researchers have used simi-
lar approaches, such as a combination ion exchange and re-
versed-phase (RP) chromatography separations, to purify
AMPs from protein extracts of plant tissue or organs [16, 25,
38]. Other combinations of chromatographic techniques have
also been utilized, including ion exchange and gel filtration
[39, 40] or RP alone [6, 41]. In each case, the purification
method used depended on the charge, size, physical stability,
and other properties of the desired peptides. We obtained 22
bound fractions using this approach; of the 6 fractions se-
lected for further analysis, 2 contained antimicrobial activity.
Electrophoretic analysis showed that fraction H11 contained
one major peptide of approximately 7 kDa and that fraction
H22 contained peptides ranging from 2 to 10 kDa Fig. (3).
Fraction H11 was subjected to C2C18 RP chromatogra-
phy, resulting in three peaks Fig. (8). The purified peptide in
peak P2 was sequenced, revealing a homology to the primary
structure of the plant defensin family of antimicrobial pep-
tides. Plant defensins are short, basic peptides composed of
45 to 54 amino acids that feature a structure of three anti-
parallel -strands and one
-helix [42]. The structure is sta-
bilized by four disulfide bonds that form a cysteine-
stabilized
 motif characteristic of these proteins [43, 44].
The presence of eight Cys residues is strictly conserved in
primary structures of plant defensins. Among the 48 amino
acid residues sequenced, Cys residues were identified at po-
sitions 3, 14, 23, 27, 38, 42, 44 and 48. The presence of eight
Cys residues was further confirmed by mass spectrometry
analysis. The only known exception to the requirement for
eight Cys residues in members of the plant defensin family is
the peptide Ph1, which has 10 Cys residues and five disulfide
bridges [45]. The comparison also indicated that among the
plant defensin family, other amino acid residues were also
conserved, such as an aromatic residue at position 10, Gly12,
Glu31 and Gly36 Fig. (9A). In the future, the peptide identi-
526 Protein & Peptide Letters, 2012, Vol. 19, No. 5
Figure 8. C2C18 reversed-phase HPLC chromatogram of fraction H11. H11 was fractionated into 4 fractions using a propanol/TFA gradient
(oblique line) and at a flow rate of 0.5 mL/min. Fractions were named as P1 to P4. Elution was monitored at 220 nm. Insert: Electrophoretic
visualization of the three bound fractions separated by C2C18 reversed-phase HPLC in tricine-SDS-PAGE. P1, P2 and P3, bound fractions
and eluted with a propanol gradient. M, molecular weight markers are indicated in kDa.
fied in this work will be referred to as ApDef1 (A. pavonina
defensin one).
Like insect and mammal defensins, plant defensins pos-
sess antimicrobial activity. In plant defensins, this activity is
directed primarily against fungal pathogens [38, 46]. The
following two yeast strains were selected for antimicrobial
activity testing in this study: S. cerevisiae, which is a model
yeast featuring the availability of myriad genetic tools, ex-
tensive knowledge of its cell physiology and broad annota-
tion of its genome [47]; and C. albicans, which is an oppor-
tunistic human pathogen causing life-threatening systemic
diseases in immunocompromised patients in whom control
of the pathogen has been difficult [48]. The fraction contain-
ing ApDef1 inhibited 83% of S. cerevisiae growth and 42%
of C. albicans growth Fig. (5).
Some plant defensins have been shown to preferentially
bind to sphingolipids in fungal membranes; Rs-AFP2 and
MsDEF1, are known to bind glucosylceramides (GlcCer),
whereas Dm-AMP1 binds mannosyldiinositol phosphorylce-
ramide (M(IP)2C) [49, 50]. Fungal membranes contain sev-
eral different classes of sphingolipids. For example, S. cere-
visiae membranes contain galactosylceramide and M(IP)2C
[51, 52] but not GlcCer, whereas C. albicans membranes do
contain GlcCer [52]. The preferential binding of plant de-
fensins to a particular sphingolipid class, together with the
differential presence of the sphingolipid classes in different
yeast membranes, may help explain why ApDef1 killed S.
cerevisiae but only inhibited the growth of C. albicans Fig.
(7).
The antimicrobial activity of AMPs is believed to be
caused by their interaction with microbial membranes. Sev-
eral AMP classes have been demonstrated to permeabilize
Soares et al.
microbial membranes, including plant defensins [32, 53],
temporins [54], plant lipid transfer protein [55] and the knot-
tin-type peptide psacotheasin [56]. Interestingly, ApDef1 did
not cause membrane permeabilization of the yeasts tested in
this study (Table 1), but the peptide killed 100% of the S.
cerevisiae cells in a viability assay Fig. (7). The results indi-
cated that fraction H11 exhibited a fungicidal effect on S.
cerevisiae and a fungistatic effect on C. albicans. In addi-
tion, a mechanism of action other than membrane permeabi-
lization may be involved because ApDef1 did not cause gross
plasma membrane disorganization, at least for S. cerevisiae.
There are several known examples of AMPs for which mem-
brane permeabilization was not the mechanism of action
responsible for microbial growth inhibition or death. These
examples include human
defenisn-2 (C. albicans) [57],
histatin-5 and (C. albicans) [58] and dermicidin-derived pep-
tides (bacteria) [59]. Aerts et al. [60] demonstrated that the
defensin from Raphanus sativus, Rs-AFP2, did not permeabi-
lize small unilamellar vesicles composed of GlcCer in the
carboxyfluorescein leakage assay. The authors also demon-
strated the production of reactive oxygen species (ROS) and
concluded that the inhibition effect was derived from an in-
tracellular mechanism.
It has been demonstrated that some plant defensins are
internalized into the fungal cytoplasm [5, 53]. For defensin
PsD1, the mechanism of fungal growth arrest appeared to be
related to the interaction of internalized defensin with intra-
cellular targets. In this case, once inside the cell, PsD1 mi-
grated to the nucleus and interacted with cyclin F, interfering
with the fungal cell cycle [5]. Other mechanisms were also
described, such as production of ROS [55, 61] and the induc-
tion of apoptosis [60].
Antimicrobial Peptides from Adenanthera pavonina Protein & Peptide Letters, 2012, Vol. 19, No. 5 527

Figure 9. Comparison of the complete amino acid sequence obtained from ApDef1 with plant defensins (A) and of the 21 amino acid N-
terminal sequence of the 6 kDa peptide from fraction H22 with ubiquitins (B). Sequences were aligned with the ClustalW [30]. I% indicates
the percentage of identical residues, which are shown in italics, and P% indicates the percentage of positive residues (representing amino
acids with the same biochemical characteristics such as charge and hydrophobicity), which are shown in gray. Gaps (-) were included to
optimize alignment. The numbers above the sequence indicate peptide length in amino acids excluding gaps, and sequences were numbered
according to ApDef1. The numbers flanking the sequences indicate the first N-terminal amino acid and the last C-terminal amino acid. The
residue E marked with an asterisk indicates the point where the two peptides were joined. Sequences from the following defensins were pre-
sented, referring to the name of the peptide or gene identification number: For (A) Cassia fistula [33], VrD2 [34], Tad1 [35], EcAMP1 [36],
Triticum aestivum (Q8L698), NsD1 [37], DmAMP1 [38], Rs-AFP1 [33], PsD2 (PSD2P81930). For (B) Oryza sativa (GI 302393766), Arabi-
dopsis thaliana (GI 302393741), Hordeum vulgare (GI 302595831), Daucus carota (GI302393742), Helianthus annuus (GI302393790) and
Schizosaccharomyces pombe (GI 302393762).

[2] Yount, N.Y.; Yeaman, M.R. Multidimensional signatures in antim-

CONCLUSION
A peptide was obtained from an A. pavonina seed extract
that exhibited fungicidal effects on S. cerevisiae and inhib-
ited the growth of C. albicans. This peptide showed similar-
ity to plant defensin peptides, both in primary sequence ho-
mology and in antimicrobial activity. This newly discovered
peptide is named ApDef1. Future studies will examine the
mechanism of action responsible for antifungal activity. In
addition, two other peptides from H22 fraction were se-
quenced. A 6 kDa peptide showed sequence homology with
ubiquitin, and a 3 kDa peptide did not exhibit homology with
any other known peptide. The importance of this discovery is
under investigation.
ACKNOWLEDGMENTS
This study comprises part of the Graduation and M.Sc.
degree dissertation of JRS, first author, and was carried out
at the Universidade Estadual do Norte Fluminense. We ac-
knowledge financial support from the Brazilian agencies
CNPq and FAPERJ. We are also thankful for financial sup-
port to Dr. Viviane V. Nascimento through a fellowship to
CAPES/PNPD.
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Received: February 1, 2011 Revised: November 22, 2011 Accepted: November 22, 2011
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