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Purification and biochemical characterization of a serine proteinase inhibitor from
Derris trifoliata Lour. seeds: Insight into structural and antimalarial features
Arindam Bhattacharyya
a,b,*
, Cherukuri R. Babu
a
a
Centre for Environmental Management of Degraded Ecosystems, School of Environmental Studies, University of Delhi, Delhi 110 007, India
b
Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India
article info
Article history:
Received 31 March 2009
Accepted 2 April 2009
Available online 4 May 2009
Keywords:
Derris trifoliata
Papilionoideae
Purification
Characterization
Conformation
Serine proteinase inhibitor
Circular Dichroism (CD)
Antimalarial
abstract
A potent serine proteinase inhibitor was isolated and characterized from the seeds of the tropical legume
liana, Derris trifoliata (DtTCI) by ammonium sulfate precipitation, ion exchange chromatography and gel
filtration chromatography. SDS–PAGE as well as MALDI-TOF analysis showed that DtTCI is a single poly-
peptide chain with a molecular mass of 20 kDa. DtTCI has three isoinhibitors (pI: 4.55, 5.34 and 5.72)
and, inhibited both trypsin and chymotrypsin in a 1:1 molar ratio. Both Dixon plots and Lineweaver–Burk
double reciprocal plots revealed a competitive inhibition of trypsin and chymotrypsin activity, with inhi-
bition constants (K
i
) of 1.7 10
10
and 1.25 10
10
M, respectively. N-terminal sequence of DtTCI
showed over 50% similarity with numerous Kunitz-type inhibitors of the Papilionoideae subfamily. High
pH amplitude and broad temperature optima were noted for DtTCI, and time course experiments indi-
cated a gradual loss in inhibitory potency on treatment with dithiothreitol (DTT). Circular Dichroism
(CD) spectrum of native DtTCI revealed an unordered structure whereas exposure to thermal-pH
extremes, DTT and guanidine hydrochloride (Gdn HCl) suggested that an abundance of b-sheets along
with intramolecular disulfide bonds provide conformational stability to the active site of DtTCI, and that
severity of denaturants cause structural modifications promoting inhibitory inactivity. Antimalarial stud-
ies of DtTCI indicate it to be a potent antiparasitic agent.
Ó2009 Elsevier Ltd. All rights reserved.
1. Introduction
Tropical rain forest legumes represent a major repository of
proteinase inhibitor (PIs) diversity, and their co-evolutionary
trends with the insect proteinases ensure a dynamic duel among
them, at the molecular level. Although PIs are believed to form a
wide-spectrum defense mechanism, they are involved in diverse
biochemical functions such as in regulation of metabolism/proteo-
lytic cascades (Shewry and Lucas, 1997), suppression of in vitro and
in vivo replication of retroviruses (Gustafson et al., 1994), hemo-
lytic activity (Tam et al., 1999), antifungal property (Swartz et al.,
1977), as anticarcinogenic agents (Kennedy, 1998) and inhibition
of intraerythrocytic development of Plasmodium falciparum (Rock-
ett et al., 1990).
PIs vary widely in their botanical origin, and in their cause and
consequence among the reproductive organs, storage organs and
vegetative tissues of most plant families. In higher plants, several
gene families have been characterized, particularly those constitut-
ing the serine protease inhibitors from Leguminosae, Solanaceae
and Graminae. Earlier studies on the distribution pattern of PIs
among seeds of leguminous trees showed an evolutionary relation-
ship between the PI family and the legume sub-families (Norioka
et al., 1988). Currently, 59 distinct families of PIs have been recog-
nized. On the basis of amino-acid sequences, localization of the
reactive sites, disulfide bridge topology, mechanism of action,
three-dimensional structure, and stability to heat and denaturing
agent, they have been placed under seven distinct families: Kunitz,
Bowman–Birk, Potato I, Potato II, Squash, Cereal and Mustard (De
Leo et al., 2002; Birk, 2003).
The two best characterized families of plant serine PIs are the
Kunitz-type and Bowman–Birk type inhibitors. Most serine PIs
are low-molecular mass molecules (3–25 kDa) that inhibit trypsin
and/or chymotrypsin. These families differ from each other in
mass, cysteine content and number of reactive sites (Richardson,
1977). Kunitz-type inhibitors are proteins of Mr 20 kDa, with
low cysteine content and a single reactive site, whereas the Bow-
man–Birk type inhibitors have Mr 8–10 kDa as well as high cys-
teine content and two reactive sites (Richardson, 1991).
Structurally, both Kunitz and Bowman–Birk inhibitors lack
a
-helix,
but vary in their mode of stability. Kunitz inhibitors are stabilized
chiefly by hydrophobic interactions of short stretches of hydrogen
bonded sheets (soybean Kunitz trypsin inhibitor) whereas the
disulfide linkages in the Bowman–Birk inhibitors minimize their
0031-9422/$ - see front matter Ó2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.04.001
*Corresponding author. Present Address: Department of Science and Technology,
Ministry of Science and Technology, Technology Bhawan, New Mehrauli Road, New
Delhi 110 016, India. Tel./fax: +91 011 26590539.
E-mail address: ady1111@rediffmail.com (A. Bhattacharyya).
Phytochemistry 70 (2009) 703–712
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Author's personal copy
conformational entropy and enhance their stability (Sweet et al.,
1974; Ramasarma et al., 1995).
The complex biology and life cycle of the apicomplexan parasite
P.falciparum has hindered attempts to control malarial infections
and also prevent transmission. Several attempts toward an effec-
tive vaccine system as well as antimalarial drugs have had only
partial success, and demands for new therapeutics have always ex-
ceeded the available treatments due to massive antigenic variation
and rapid drug resistive prowess of the parasite. In spite of these
limitations, regular updates on the parasite genome biology pro-
vide a platform to renew our efforts towards complete control of
malarial infection. In fact, a recent survey of the parasite genome
has revealed that over 90 putative proteases belonging to 26 fam-
ilies of five clans constitute the parasite genome (Wu et al., 1992).
These proteases (11% aspartic, 36% cysteine, 22% metallo, 17% ser-
ine and 14% threonine) are engaged in a functionally diverse but
well orchestrated cascade of proteolytic processes throughout its
life cycle with particular emphasis in host cell invasion, nutrition
and growth, and the processing of precursor proteins (Klemba
and Goldberg, 2002; Pattanaik et al., 2003). Thus, a renewed ap-
proach towards the discovery of new antiparasitic molecules has
been the design and discovery of different types of PIs towards
these parasite proteases, as PIs of different classes have been
shown to arrest the parasite invasion of erythrocyte and prevent
its survival inside the host (Blackman, 2000; Rosenthal, 2002).
Co-evolutionary trade-offs, which are the precursors of a pleth-
ora of plant–herbivore interactions in tropical forests, engenders a
vast reservoir of PIs in leguminous plants, some of which may
prove to be effective antimalarial chemotherapeutics. Moreover,
not much attempt has been done on plant derived PIs as antimal-
arials and even the limited studies conducted (Dejkriengkraikhul
and Wilairat, 1983) have yielded only partial success. Thus, in light
of the aforesaid facts, we report here the purification, structure–
function characterization and antiplasmodial property of a serine
PI from the seeds of Derris trifoliata (DtTCI), a commonly occurring
liana along mangrove and coastal forests of Great Nicobar Island,
Andaman and Nicobar Islands, India.
2. Result and discussion
2.1. Purification of the Derris seed proteinase inhibitor
A crude inhibitor (CI) preparation was obtained by saline
extraction of ground seeds of D.trifoliata showing 53% and 58%
inhibition of trypsin and chymotrypsin activity, respectively.
Table 1 demonstrates the inhibitor characteristics after (NH
4
)
2
SO
4
precipitation, DEAE 52 cellulose ion exchange chromatography, gel
filtration chromatography on Superdex S-75 and Q-Sepharose ion
exchange chromatography. DEAE ion exchange chromatography
of the 0–75% (NH
4
)
2
SO
4
precipitated inhibitor revealed four major
protein peaks, each possessing either antitrypsin or antichymo-
trypsin activity (Fig. 1a). The protein eluted with 0.2 M NaCl
(fractions: 82–100 ml) demonstrated a strong trypsin inhibitory
(11.9 TIU/A
280
) as well as chymotrypsin inhibitory (12.2
CIU/ A
280
) activity (Table 1), and was subsequently applied on a
Superdex S-75 column. The elution pattern showed a major leading
peak (DtTCI-1) followed by a minor trailing peak (DtTCI-2)
(Fig. 1b). Expectedly, DtTCI-1 inactivated over 90% of both trypsin
and chymotrypsin activity, with similar specific activities towards
both the serine proteases. DtTCI-1 was rechromatographed on
Q-Sepharose which further resolved into two main peaks (P2 and
P3) (Fig. 1c). Purified DtTCI (P2) represented 0.1% of the total seed
proteins of D.trifoliata. These results concur well with previously
studied Papilionoid inhibitors (Haq and Khan, 2003; Garcia et al.,
2004).
2.2. Molecular mass evaluation and isoinhibitor forms
Analyses of purified DtTCI by SDS–PAGE gel electrophoresis
both in presence as well as absence of DTT yielded a single poly-
peptide chain corresponding to a molecular weight of 20 kDa
(Fig. 1c). In addition, MALDI-TOF analyses also showed a molecular
mass of 20.1 kDa (Fig. 2a). Studies conducted previously on Kunitz
inhibitors belonging Papilionoideae sub-family (Garcia et al., 2004;
Gomes et al., 2005), have also reported similar results. Isoelectric
focusing of DtTCI revealed the acidic nature of the inhibitor and,
also the presence of three isoinhibitors (Fig. 2b). The three isoin-
hibitors had pIof 4.55, 5.34 and 5.72, respectively. Similar isoinhib-
itor forms and a strong acidic trait are typical attributes of several
Kunitz PIs (Richardson, 1991). Physiologically, occurrence of such
isoinhibitors may be a part of the survival strategies evolved by
the host plant.
2.3. N-terminal amino acid sequence analysis
The N-terminal sequence analysis of DtTCI exhibited a strong
similarity with other members of the Kunitz PI from the Papilionoi-
deae subfamily (Onesti et al., 1991; Kouzuma et al., 1992; Terada
et al., 1994; Datta et al., 2001). In fact, DtTCI showed over 70% se-
quence similarity with PIs of Erythrina variegata and Canavalia line-
ata and close resemblance to trypsin inhibitors from Psophocarpus
tetragonolobus and Erythrina caffra (Fig. 3a).
Table 1
Summary of the purification of a serine proteinase inhibitor from Derris trifoliata (DtTCI).
Step/characteristics Total protein (mg) % Inhibition Specific activity % Recovery Purification factor
T
a
C
b
T(TIU/ A
280
)
c
C(CIU/ A
280
)
d
TC
Crude inhibitor 632.5 53.3 57.8 1.1 1.3 100 1.0 1.0
Ammonium sulfate precipitation 0–75% 161.0 75.1 68.3 3.0 3.6 25.5 2.7 2.8
DEAE 52 cellulose chromatography NaCl (M) 0.1 33.3 76.7 87.5 5.2 5.5 5.3 4.7 4.2
0.2 26.8 82.2 89.2 11.9 12.2 4.2 10.8 9.4
0.3 15.8 78.4 70.1 10.2 10 2.5 9.1 7.7
0.4 7.1 53.8 31.6 9.2 2.6 1.1 8.4 2.0
Superdex S-75 gel filtration 6.3 92.3 97.6 56.5 58.6 0. 7 51.4 45.1
Q sepharose chromatography NaCl (M) 0.2 0.8 94.7 98.2 70.8 71.1 0.1 64.4 54.7
0.3 1.5 79.6 82.5 12.5 11.8 0.2 11.4 9.1
Note: One trypsin or chymotrypsin unit is defined as 1
l
mol of substrate hydrolyzed per minute of reaction. One inhibition unit is defined as unit of enzyme inhibited. Specific
activity is defined as inhibition units per absorbance unit, at 280 nm, of the inhibitor.
a
Trypsin.
b
Chymotrypsin.
c
Trypsin inhibitory units.
d
Chymotrypsin inhibitory units.
704 A. Bhattacharyya, C.R. Babu / Phytochemistry 70 (2009) 703–712
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2.4. Inhibitor kinetics and inhibition constant
DtTCI suppressed both tryptic as well as chymotryptic activity
with similar potency. The titration profiles for both trypsin and
chymotrypsin remained linear up to 90% inhibition and extrapo-
lation of the curve engendered a 1:1 trypsin/chymotrypsin–DtTCI
complex (Fig. 3b).
Predictably, kinetic studies on the initial rates of reaction in the
presence or absence of DtTCI followed the Michaelis–Menten
equation, and both the Lineweaver–Burk double reciprocal plots
(a)
(b)
(c)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64
Fraction number
Absorbance at 280 nm
0
10
20
30
40
50
60
70
80
90
100
% inhibition
P3
P2
P1
NaCl (M)
0.6
0.5
0.4
0.3
0.2
0.1
66.0
20.1
14.4
M P2 P3
R NR
1 2 3 4
Fig. 1. (a) Ion exchange chromatography of the 0–75% ammonium sulfate fractionated proteins of D.trifoliata seed extract, on DE 52-Cellulose. Column dimensions:
2.5 18 cm; Starting buffer: 20 mM Tris HCl (pH 8). The line cutting across the chromatogram indicates application of a step gradient in the NaCl concentration in the starting
buffer. (b) Size-exclusion chromatography of 0.2 M NaCl DtTCI fraction (eluted from the DE-52 Cellulose column) onto a Superdex S-75 column. Inset: SDS–PAGE profile of
DtTCI-1. (c) Elution profile of DtTCI-1 on Q-Sepharose chromatography column (2.5 17 cm) equilibrated with 20 mM Tris HCl (pH 8). Protein elution was performed using a
linear gradient (0.1–0.5 M) of NaCl. Fractions (3 mL) were collected and assayed for antitryptic (
D
) and antichymotryptic (h) activities. Inset: SDS–PAGE of 0.2 M (lanes 2 and
3: under reducing (R) and non-reducing (NR) conditions, respectively) and 0.3 M (lane 4) NaCl fractions of DtTCI following re-ion exchange chromatography on Q-Sepharose.
Lane 1: protein molecular weight marker.
A. Bhattacharyya, C.R. Babu / Phytochemistry 70 (2009) 703–712 705
Author's personal copy
(Fig. 4a and c) and Dixon plots (Fig. 4b and d) indicated a compet-
itive inhibition of both trypsin and chymotrypsin activity, respec-
tively. The inhibition constants (K
i
) of DtTCI against both trypsin
(TAME hydrolyzing units) and chymotrypsin (BTEE hydrolyzing
units) activity were 1.7 10
10
and 1.25 10
10
M, respec-
tively (Fig. 4b and d). Similar K
i
values were also recorded for PIs
isolated from other legumes (Chaudhary et al., 2008; Bhattachar-
yya et al., 2006; Garcia et al., 2004).
2.5. Stability attributes
DtTCI showed 80% and over 60% inhibition of trypsin/chymo-
trypsin activity on a temperature gradient of 15–75 °C and over a
pH range of 2–12 (data not shown), respectively. Interestingly,
DtTCI retained over a 2-fold higher trypsin/chymotrypsin inhibi-
tion following exposure to 6 N HCl as compared to conditions of
1 N NaOH (Table 2). Earlier studies on many of the Kunitz PIs have
also reported similar defiance to extremes of thermo-pH condi-
tions and have attributed this stability to the apparent ability of
the inhibitor to reversible denaturation through a transient inter-
mediate, presence of aromatic (tyrosine) residues generally impli-
cated in energy transfer and disulfide bonds (Roychaudhuri et al.,
2003; Liao et al., 2007; Yoshizaki et al., 2007; Zhou et al., 2008;
Chaudhary et al., 2008). The effect of DTT on the inhibitory activity
of DtTCI was also examined. DtTCI was also able to withstand
reducing conditions of 10 mM DTT for up to 2 h following which
there was a steady loss of inhibitory activity with less than 10%
inhibitory activity at the end of 5 h (Table 2;Fig. 5a). Similar effect
of DTT was also observed for other Kunitz PIs (Garcia et al., 2004;
Lingaraju and Gowda, 2008; Zhou et al., 2008). Although intramo-
lecular disulfide linkages have been ascribed to be the prime factor
behind the functional stability of the inhibitor against denaturants
(Zhou et al., 2008; Liao et al., 2007; Macedo et al., 2007; Oliveira
et al., 2007), additional factors such as placement/arrangement of
the disulfide linkages (buried, rather than surface exposed) within
the molecule are also critical for the overall stability of the inhibi-
tor (Tetenbaum and Miller, 2001; Yoshizaki et al., 2007; Macedo
et al., 2007).
2.6. CD based conformation study
Estimation of the secondary structure of native DtTCI as well as
DtTCI exposed to extreme heat, pH, 6 N guanidine hydrochloride
and DTT in solution was carried out using near and far-ultraviolet
CD based studies. The far-UV CD spectrum of native DtTCI (Fig. 5b)
was similar to that of b-II proteins, exhibiting a sharp minimum
ellipticity at 200 nm and a positive band at 192 nm with a minor
shoulder close to 230 nm. The detailed structural composition of
the protein showed a large fraction (40%) of b-sheets (Table 2).
Similar CD spectrums were also noted for several other legume
PIs (Liao et al., 2007; Yoshizaki et al., 2007; Bhattacharyya and
Babu, 2006; Araujo et al., 2005).
CD-based conformational evaluation of DtTCI following expo-
sure to severe heat and acid–alkali conditions showed gross devi-
ations among the
a
-helix and b-sheets content which were in
concurrence with the reduced inhibitory activity (Table 2; spec-
trum not shown). The results are in agreement with experimental
studies carried out on other Kunitz PIs (Liao et al., 2007; Yoshizaki
(b)
8.65
8.45
8.15
7.35
6.85
6.55
5.85
5.20
4.55
3.50
DtTCI 1 (5.72)
DtTCI 2 (5.34)
DtTCI 3 (4.55)
(a)
Fig. 2. (a) MALDI-TOF mass spectrum of DtTCI. (b) Isoelectric focusing of DtTCI on
Ampholine polyacrylamide gel plates (pH range 3.5–9.5, Pharmacia) over a broad-
range (pI: 3–10) calibration kit. Lane 1: marker and lane 2: purified DtTCI showing
three isoinhibitors with pIat 5.72, 5.34 and 4.55.
(a)
(b)
0
10
20
30
40
50
60
70
80
90
100
00.511.52
Molar ratio (inhibitor: enzyme )
(%) Residual enzyme activity
Tr yp s in
Chymotryps in
% Alignment
Score
DtTCI ELVLDVNGDQVRNGGTYYL
EvTI ELV-DVEGEDVVNGGTYYM
ClTI --VLDTDGDMVRNGGIYYI
PtTI EELVDVEGKTVRNGGTYYL
EcTI -VLLDGNGEVVQNGGTYYL
BvTI EIVLDQNGNPVRNSGGRYY
STI DFVLDNEGNPLSNGGTYYI
Sporamin A TPVLDINGDEVRA-GGNYY
AcTI -KELLDADGDILRNGGAYY
CaTI EQVLDTNGNPLIPGDEYYI
ElTI -VLLDGNGEVVQN-GGIYY
PpTI -APLEDSLAAKLNNGTSYY
72
70
68
66
57
52
52
52
47
44
31
Fig. 3. (a) N-terminal sequence alignment of related Kunitz Proteinase Inhibitors
from Erythryna variegata,Canavalia lineata,Psophocarpus tetragonolobus,Erythryna
caffra,Bauhinia variegata,Glycine max,Ipomoea batatas,Acacia confusa,Cicer
arietinum,Erythryna latissima,Poecilanthe parviflora. (b) Titration curves of bovine
trypsin and
a
-chymotrypsin inhibition by DtTCI. Following an incubation of 5 min,
percent residual trypsin (——) and chymotrypsin (—j—) activities were measured
at 25 °C (at pH 8.1) using substrates TAME and BTEE, respectively, in the presence of
varying concentrations of DtTCI.
706 A. Bhattacharyya, C.R. Babu / Phytochemistry 70 (2009) 703–712
Author's personal copy
et al., 2007; Garcia et al., 2004; Haq and Khan, 2003; Macedo et al.,
2003). To examine the changes accorded by DtTCI at a structural
level following DTT exposure, far-UV CD measurements were per-
formed. Noticeably, marked differences in the far-UV spectrum of
native and reduced DtTCI were recorded which included a loss in
the negative band at 200 nm in the latter (Fig. 5c). Also, a minor
reduction in ellipticity of the 230 nm band was noted, which might
have been due to the chirality of the cysteine bonds. Table 2 shows
a loss in the b-sheets and a concomitant increase in the helical con-
tent of the inhibitor. Conformational changes in DtTCI due to disul-
fide bond reduction disrupted the protein stability, and partly
affected the reactive site, as evidenced by the retention of 20%
trypsin and 40% chymotrypsin inhibitory activity, respectively
after 3 h of reduction by 10 mM DTT (Fig. 5a). Similar partial pro-
tection of the active site has also been observed for a Kunitz PI from
Entada scandens (Lingaraju and Gowda, 2008). It is interesting to
note, that under extreme DTT conditions the magnitude of loss in
inhibitory activity of Kunitz PIs varies from species to species.
For instance, upon exposure to 100 mM DTT, the inhibitory activi-
ties of some of the Kunitz PIs were abolished (Garcia et al., 2004;
Macedo et al., 2003) whereas some others remained completely
unaffected (Chaudhary et al., 2008; Lehle et al., 1996). It is pre-
sumed that the intramolecular disulfide bonds and their distance
from the reactive site residues determine the functional stability
of these inhibitors in presence of denaturing agents (Garcia et al.,
2004; Lehle et al., 1996). However, studies on the susceptible (sin-
gle polypeptide) soybean trypsin inhibitor (Tetenbaum and Miller,
2001) or stable Bauhinia species inhibitors (devoid of any disulfide
linkages), indicated that intramolecular disulfide bonds in Kunitz-
type PIs may be essential, but not exclusive to the stabilization of
the reactive site loop.
Upon exposure to 6 N Gdn HCl for 2 h at 70 °C, DtTCI showed
gross conformational perturbations due to a total denaturation of
the protein (Fig. 6a and 6b). In fact, the near UV spectrum recorded
a significant decrease in amplitude for the broad negative elliptic-
ity band between 290 and 250 nm in comparison to the native
(a) (b)
(c) (d)
0
5
10
15
20
25
30
35
40
45
50
-3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11
DtTCI [x10-1 nM]
DtTCI [x10-1 nM]
BTEE (0.535 mM)
BTEE (1.07 mM)
0
3
6
9
12
15
18
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5
TAME [0.005 mM]
TAME [0.01 mM]
0
3
6
9
12
15
18
-200 -100 0 100 200
1/ [s] (TAME) mM
-1
1/ v (OD
247
mM/min /mL)
-1
1/ v (OD
247
mM/min /mL)
-1
[I] = 0 nM
[I] = 5 nM
[I] = 6.6 nM
[I] = 10 nM
Ki
Ki
0
4
8
12
16
20
24
-2 -1 0 1 2
1/ [s] (BTEE) mM) -1
1/v (OD
256
mM/min/m L)
-1
1/v (OD
256
mM/min/mL)
-1
[I] = 10 nM
[I] = 6.6 nM
[I] = 5 nM
[I] = 0 nM
Fig. 4. Kinetic analysis of trypsin and chymotrypsin inhibition by purified DtTCI. A Lineweaver–Burk plot showing competitive nature of trypsin (a) and chymotrypsin (c)
inhibition by DtTCI and Dixon plot for the determination of the inhibition constant (K
i
) of DtTCI towards trypsin (b) and chymotrypsin (d) at two different concentrations of
TAME and BTEE, respectively. The reciprocals of velocity were plotted against variable DtTCI concentration.
Table 2
CD secondary structure content and serine proteinase inhibitory activity of native and treated DtTCI. All experiments were performed in 10 mM Tris buffer/50 mM NaCl, pH 8, at
25 °C.
DtTCI (D) CD secondary structure content (%) % Inhibition
a
-Helix b-Sheet Unordered T
a
C
b
D only 4 39 57 91.3 ± 6.1 95.5 ± 6.8
D (100 °C; 2 h) 21 4 75 36.4 ± 3.2 32.6 ± 4.5
D + 6 N HCl (30 °C; 2 h) 8 30 62 72.5 ± 6.2 88.2 ± 5.7
D + 1 N NaOH (30 °C; 2 h) 19 21 60 31.4 ± 4.7 28.8 ± 4.4
D + Gdn HCl (6 N; 40 °C; 5 h) 10 19 71 19.8 ± 2.5 26.2 ± 2.8
D + Gdn HCl (6 N; 70 °C; 2 h) 23 4 73 4.2 ± 0.6 8.6 ± 0.8
D + DTT (10 mM; 37 °C; 2 h) 14 26 60 62.4 ± 5.1 73.4 ± 5.7
D + DTT (10 mM; 37 °C; 5 h) 30 8 62 5.8 ± 0.4 9.2 ± 0.6
a
Trypsin.
b
Chymotrypsin.
A. Bhattacharyya, C.R. Babu / Phytochemistry 70 (2009) 703–712 707
Author's personal copy
DtTCI (Fig. 6a). Detailed examination revealed that the total loss of
secondary structure was primarily due to a complete conversion of
the b-sheets to helical and unordered contents (Table 2). The se-
vere effect of Gdn HCl on DtTCI is in accordance with similar stud-
ies on other Kunitz PIs (Souza et al., 2000; Haq and Khan, 2003;
Leach and Fish, 1977).
2.7. Antimalarial activity
The in vitro antimalarial efficacy of DtTCI was propounded by
monitoring a decrease in the pLDH (parasite Lactate Dehydroge-
nase) activity against the two P. falciparum lines, 3D7 and FCR3.
Typically, a dose-dependent curve was observed against both the
parasite lines although marked differences in the erythrocyte inva-
sion inhibitory levels existed between them (Fig. 7a). In fact, DtTCI
showed a minor fold (1.35 times) higher inhibitory capacity
against 3D7 as compared to FCR3. The IC
50
values obtained for
DtTCI for P.falciparum 3D7 and FCR3 were 9 and 16.35
l
M, respec-
tively (Fig. 7c).
A few studies prior to our study involving the role of plant de-
rived serine PIs didn’t hold much promise (Dejkriengkraikhul and
Wilairat, 1983) and consequently, efforts to explore plant PIs as
effective antimalarial drugs were also limited. However, some re-
cent studies have advocated that proteolytic processing by serine
proteases is critical for parasite invasion of erythrocytes. For
example, Kato et al. (2005) have suggested that Apical Merozoite
Antigen 1 (AMA), an indispensable parasite protein, plays an
important but not exclusive role in invasion of human erythro-
cytes through a process that involves processing of the erythro-
cyte surface protein Kx, by a trypsin-like parasite protease.
Similarly, a recent study has also reported that secondary prote-
olytic processing of yet another vital parasite surface protein –
the Merozoite Surface Protein 7 (MSP7), is abrogated in the pres-
ence of serine protease inhibitors (Pachebat et al., 2007). Further
evidences on the role of serine proteases particularly that of chy-
motrypsin type in parasite invasion of erythrocyte have been doc-
umented by earlier studies using chymostatin as an
antiplasmodial agent (Howell et al., 2003; Dutta et al., 2003,
2005).
Our data exhibits a reduction in parasitemia following exposure
to DtTCI and this may be due to an effective blocking of the essen-
tial serine proteolytic activity of/on some major surface antigens of
the parasite. Although, several studies conducted earlier have doc-
umented PIs belonging to serine, cysteine, aspartyl and metallo-
protease classes as successful antimalarial agents (Blackman,
2000; Rosenthal, 2002), much work towards understanding the
intricate mechanisms of parasite protease–protease inhibitor
interactions still remains and need to be resolved systematically.
Moreover, ever since Wu et al. (1992) demonstrated that the gen-
ome of P.falciparum houses several classes of stage-specific prote-
ases each of which has some functional connotation, the relevance
of these molecules as effective targets for antimalarial drugs have
been recognized and renewed efforts have been galvanized in that
(a)
(b)
(c)
0
20
40
60
80
100
0 30 60 90 120 150 180 210 240 270 300
Time (Min)
% Residual inhibitory activity
% Chymotrypsin
% Trypsin
Fig. 5. Conformational stability and antiproteolytic activity of DtTCI on exposure to
DTT. Both the residual trypsin and chymotrypsin inhibitory activity were assayed by
using substrates TAME and BTEE, respectively, in 50 mM Tris–HCl, pH 8.0. All
experiments were performed in 10 mM Tris buffer/50 mM NaCl, pH 8. Each point is
the mean of three assays. (a) Time course profile of the antiproteolytic activity of
DtTCI after incubation with 10 mM DTT at 37 °C. (b) Far UV CD spectra of native
DtTCI. (c) Far-UV CD spectra of native DtTCI and DTT reduced with DtTCI (37 °C; 5 h).
Fig. 6. Effect of 6N Gdn HCl on conformational stability of DtTCI (a) Near UV CD
spectra at various temperatures (b) Far UV CD profile following treatment with 6N
Gdn HCl at 70 °C for 2 h. Inhibitor concentration of 0.5 mg/ml in 10 mM Tris buffer/
50 mM NaCl, pH 8 was used. Each point is the mean of three assays.
708 A. Bhattacharyya, C.R. Babu / Phytochemistry 70 (2009) 703–712
Author's personal copy
direction. Consequently, the role of plant serine PIs needs to be re-
explored as effective agents against the malarial parasite as well as
the spread of malarial disease.
3. Materials and methods
3.1. Plant material
Derris trifoliata Lour. seeds were manually harvested from the
coastal/mangrove forests of Great Nicobar Island, Andaman and
Nicobar Islands, India. Seeds were air-dried and stored at 20 °C
before use.
3.2. Isolation and purification of DtTCI
Fifty gram of D.trifoliata seeds were soaked overnight in 0.15 N
NaCl. The swollen cotyledons were then homogenized with 350 ml
of saline Tris buffer (20 mM Tris, pH 8.0; 0.15 M NaCl) containing
1 mM sodium metabisulfite at 4 °C. Following filtration through a
chilled 4-fold muslin cloth, the homogenate was centrifuged for
15 min at 12,000 gat 4 °C. The supernatant (crude extract) ob-
tained was fractionated by a 0–75% (NH
4
)
2
SO
4
precipitation. Sub-
sequently, the precipitated protein was dialyzed against cold
deionized water, lyophilized and submitted to proteinase inhibi-
tory assays. The dialyzed protein showing high antitryptic activity
was applied to an anion exchanger column DEAE 52, pre-equili-
brated with 20 mM Tris buffer, pH 8.0. Protein fractions (5 ml)
were eluted using a step gradient (0.1–0.5 M) of NaCl at a flow rate
of 0.5 ml/min. Fractions eluted with 0.2 M NaCl were pooled, dia-
lyzed, lyophilized and submitted (1.0 mg/ml) to a Superdex S-75
column pre-equilibrated with 0.1 M Tris–HCl buffer (pH 8.0) con-
taining 0.1 M NaCl, for gel filtration chromatography. DtTCI active
fractions (0.5 ml; flow rate: 20 ml/h) were collected. The column
fractions with DtTCI activity were dialyzed, concentrated and
loaded onto a strong anion exchanger column Q-Sepharose Fast
Flow pre-equilibrated with 50 mM Tris–HCl buffer (pH 8.0),
50 mM NaCl. The retained proteins were eluted with 50 mM
Tris–HCl (pH 8.0) containing 1 M NaCl at flow rate of 1 ml/min
and dialyzed against cold deionized water. The antitryptic peak
(P2) was pooled and concentrated for further analyses.
3.3. Proteinase inhibition studies
Following Birk (1976), trypsin and chymotrypsin inhibitory as-
says were carried out using the substrates TAME (tosyl–arginyl–
methyl ester hydrochloride) and BTEE (n-benzoyl tyrosine ethyl es-
ter) respectively. The remaining esterolytic activity of trypsin and
chymotrypsin towards their respective substrates in presence of
DtTCI were estimated. Both the proteinases were pre-incubated
with DtTCI for 5 min at 25 °C, in 1 ml Tris buffer (46 mM) contain-
ing 11.5 mM CaCl
2
, pH 8.1, prior to the reaction. One trypsin or
chymotrypsin unit is defined as 1
l
mol of substrate hydrolyzed
per minute of reaction. One inhibition unit is defined as unit of en-
zyme inhibited. Specific activity is defined as trypsin inhibition
units (TIU) per absorbance unit, at 280 nm (A
280
), of the inhibitor.
3.4. Estimation of proteins
Protein content was measured by Coomassie blue staining
according to the procedure of Bradford (1976) as well as from
the absorbance at 280 nm. BSA (1 mg/ml) was used as a protein
standard.
3.5. Reduction and S-alkylation
As outlined by Crestfield et al. (1963), DtTCI (5 mg) was reduced
with 0.1 M dithiothreitol and S-carboxymethylated using iodoace-
tic acid. Subsequent to desalting, DtTCI was redissolved in 0.05 M
Tris buffer pH 8.0, and gel filtrated on Superdex S-75, equilibrated
with the same buffer.
3.6. SDS–PAGE profiles
Following Laemmli (1970), purified DtTCI was subjected to
SDS–PAGE using 5–15% polyacrylamide slab gel system, after pre-
treating the protein sample with Laemmli’s buffer. The gels were
stained by Coomassie brilliant blue R250. A low molecular weight
range (14–116 kDa) of protein molecular weight markers (Amer-
sham Biosciences), were used.
(a)
(b)
(c)
0
10
20
30
40
50
60
70
80
Proteinase Inhibitor (
µ
M)
% Erythrocyte invasion inhibition
DtTCI
Chymostatin
DtTCI
0 0 1.3 3.42 6.17 9.83 14.9 35.4 48.2 69.5
Chymostatin
0 0 0.08 0.48 2.67 6.78 11.3 26.8 36.6 52.2
0 0.03 0.06 0.13 0.25 0.5 1 5 10 25
0
10
20
30
40
50
60
70
80
Proteinase Inhibitor (
µ
M)
% Erythrocyte invasion inhibition
DtTCI
Chymostatin
DtTCI
0 0 1.12 2.04 3.46 5.76 10.1 30 40.2 51.3
Chymostatin
0 0 0.03 0.21 0.88 2.05 4.73 17.2 26.5 33.7
0 0.03 0.06 0.13 0.25 0.5 1 5 10 25
Fig. 7. LDH based growth inhibition assay of DtTCI and chymostatin on the growth
of P.falciparum strains 3D7 (a) and FCR3 (b) Mature trophozoite enriched parasite
cultures (0.3 ± 0.1%; 2% hematocrit) were incubated with DtTCI for 42 h at 37 °C and
examined for inhibition of erythrocyte invasion. Each point represents the
mean ± S.E. from one representative experiment in triplicate. (c) In vitro efficacy
of DtTCI on inhibition of P.falciparum (3D7 and FCR3 strains) growth. GraphPad
Prism (5.1 program) was employed to generate the inhibition curve. Each point
represents mean ± SE of three LDH based GIA assays.
A. Bhattacharyya, C.R. Babu / Phytochemistry 70 (2009) 703–712 709
Author's personal copy
3.7. Mass spectrometry
Matrix-assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF/MS) was used to analyze the purity
and absolute molecular mass of DtTCI using a Bruker Daltonics
Ò
model Ultraflex spectrometer. Spectra were recorded in the posi-
tive-ion mode at 50 Hz laser frequency. Samples were prepared
by mixing equal volumes of sinapinic acid in 0.1% TFA (aq.), aceto-
nitrile (2:1), and the inhibitor solution. From this mixture, 1
l
l was
spotted on to the sample plate and allowed to evaporate to dry-
ness. Bovine trypsinogen was used for internal calibration.
3.8. N-terminal sequencing
The first 20 amino acids from the N-terminal end were se-
quenced by Edman degradation method on an Applied Biosystem
Procise Sequencer. Percentage sequence identity with trypsin
inhibitors from other members of Mimosoideae sub-family was
determined using BCM search launcher for multiple sequence
alignment (Worley et al., 1998) and BOXSHADE 3.21 was used
for shading of multiple alignment files.
3.9. Isoelectric focusing
Isoelectric focusing was performed using a Phast System appa-
ratus from Pharmacia LKB Biotechnology to separate the iso-inhib-
itors of DtTCI. Phast Gel IEF 3–9, which operates in the pH range 3–
9, and Ampholine polyacrylamide gel plates from Pharmacia (pH
range 3.5–9.5) were used together with a Pharmacia broad-range
pIcalibration kit containing proteins with various isoelectric points
ranging from 3 to 10.
3.10. Kinetic analysis of DtTCI
Kinetic analyses of DtTCI activity were carried out by using Dix-
on plot estimation (Segel, 1975). A series of DtTCI concentrations
(nM) of 4, 5, 6.6 and 10 were used to determine the inhibition con-
stant (K
i
). Enzyme inhibition was characterized at two different
substrate concentrations [TAME (0.005 and 0.01 mM) and BTEE
(0.53 and 1.07 mM)], for trypsin and chymotrypsin, respectively.
The initial slope
v
was determined for each inhibitor concentration.
The reciprocal velocity (1/
v
) versus [PI], for each substrate concen-
tration, [S
1
] and [S
2
], was plotted. A single regression line for each
[S] was obtained, and the K
i
was calculated from the intersection of
the two lines. The velocity of the reaction was expressed as 1/v
(OD
247
mM/min/ml)
1
. The mechanism of inhibition was deter-
mined using Lineweaver–Burk plots, in which the inverse of the
initial rate was plotted against the inverse of the substrate concen-
tration in the absence or presence of DtTCI.
3.11. Biological properties of DtTCI
The temperature optima of purified DtTCI was estimated by its
trypsin and chymotrypsin inhibitory activity, under varying tem-
peratures (5–100 °C, with an increment of 10 °C) in 0.05 M Tris–
HCl buffer, pH 8.0. For each temperature, an incubation period of
60 min duration was maintained. Both antitrypsin and antichymo-
trypsin assays under a range of pH (2–12) conditions were used to
assess the pH optima of DtTCI. Purified DtTCI was mixed with buf-
fers of different pH respectively in the ratio of 1:1 (v/v), and incu-
bated for 3 h. The buffers used were 0.02 M each of glycine–HCl
(pH 2.0–3.5), sodium phosphate (pH 6–7.5), Tris–HCl (pH 8.0–
10.0) and glycine–NaOH (pH 10.5–13.0). Three replicates were
maintained for each temperature and pH condition. In addition, ef-
fects of extreme acidic and basic conditions on the inhibitory
capacity of DtTCI were examined by treatment of the inhibitor
with 6 N HCl and 1 N NaOH. Further, DtTCI was (0.5 mg/ml) was
incubated with dithiothreitol (DTT) at final concentrations of
2 mM and 10 mM for 30–300 min each at 37 °C. After the stipu-
lated time period the reaction was terminated using 4 mM of iodo-
acetamide, and antitryptic activity of DtTCI was assessed.
Replicates in triplicate were maintained for the different chemical
treatments.
3.12. Circular Dichroism (CD) of DtTCI
Different CD spectral measurements were taken for native
DtTCI, and DtTCI exposed to (i) thermo-pH extremities and (ii)
treated with 6 M guanidine (Gdn) HCl and DTT over the range of
190–250 nm (protein solutions of 0.5 mg/ml) following Leach and
Fish (1977). The CD spectra were collected on a Jasco spectropolar-
imeter (J-810 Circular Dichroism System) equipped with a stopped
flow chamber and thermostated cell holder with 1 nm bandwidth,
a scanning speed of 50 nm/min at cell length of 0.2 cm and at 25 °C
temperature (over three accumulations) in 10 mM Tris/HCl, pH 8
(Bhattacharyya and Babu, 2006). The molar ellipticity was obtained
and the percent secondary structure was predicted using the pro-
gram k2d (Yang et al., 1986; Andrade et al., 1993).
3.13. Parasite culture
P. falciparum lines 3D7 and FCR3 cultures were maintained and
synchronized in vitro using the method of Trager and Jensen
(1976). The cultures of P. falciparum were maintained with human
O
+
RBCs suspended in 1complete culture medium [RPMI 1640
containing 0.5% AlbuMAX-I (Invitrogen) and 0.2% sodium bicar-
bonate, gentamicin (25 mg/l) and hypoxanthine 0.4 mM]. The
two culture lines of P. falciparum cultures were synchronized twice
by sorbitol treatment, and late stage trophozoites were enriched by
flotation on 65% percoll, respectively, as outlined by Kennedy et al.
(2002). The parasitemia of the mature trophozoite enriched prepa-
rations were determined by Giemsa staining and preparations of
over 90% purity were used in the erythrocyte invasion inhibition
assay.
3.14. Growth inhibition assay (GIA)
Following enumeration of parasitemia using Giemsa, the ongo-
ing culture was diluted using 2complete culture medium (dou-
ble concentration of the basic constituents of the 1culture
medium) to a final parasitemia of 0.3 ± 0.1% with a 2% hematocrit
for the assay. The assays were conducted in 96-well plates. DtTCI
was serially diluted in PBS, and 50
l
l was added to sterile flat-bot-
tomed microtitre wells (Nunc). Chymostatin, a chymotrypsin like
serine proteinase inhibitor was used as a positive control following
Dutta et al. (2003). To each well, 50
l
l of synchronized, mature tro-
phozoite-stage parasites were added (2% hematocrit, 0.3% parasite-
mia). Uninfected RBC controls (Blank) and negative controls (only
PBS, without DtTCI) were also included in the assay. The assay mix-
ture was incubated for 42 h at 37 °C in a moist atmosphere of 94%
N
2
,1%O
2
and 5% CO
2
. A 50-
l
l volume of parasites was then
washed with 250
l
l ice-cold PBS following which, 240
l
l of super-
natant was aspirated out of each well and the plate was frozen at
20 °C for at least 2 h before performing LDH assay for determina-
tion of relative parasitemia levels.
3.15. Lactate dehydrogenase (LDH) assay
Following thawing of the frozen plates, relative parasitemia lev-
els were determined by assaying for parasite lactate dehydroge-
nase activity (Basco et al., 1995; Kennedy et al., 2002). Briefly,
immediately prior to use, 100
l
l of lactate dehydrogenase assay
710 A. Bhattacharyya, C.R. Babu / Phytochemistry 70 (2009) 703–712
Author's personal copy
buffer (0.1 M Tris [pH 8.0] containing 50 mM sodium
L
-lactate,
0.25% Triton X-100, 5
l
g of 3-acetylpyridine adenine dinucleotide,
0.1 units of diaphorase and 20
l
g of Nitro Blue Tetrazolium) (all re-
agents were from Sigma) was added to each well. After addition,
the plate was instantly spun (5–15 s) at 1800gto eliminate bub-
bles. Next the plate was incubated for 30 min in the dark at room
temperature, with shaking. Absorbance was measured at 650 nm,
and percent inhibition of invasion was calculated as follows:
Assays were done in triplicate, and error bars represent the
standard deviations.
%Inhibition ¼100%
A
650
DtTCI treated sample A
650
RBC only 100
A
650
infected RBC A
650
RBC only
:
3.16. Statistical analysis
For the pLDH based assays, all values were normalized to per-
cent control activity and 50% inhibitory concentrations (IC
50
s) were
calculated by non-linear regression with the Prism 5.1 program
(GraphPad Software).
Acknowledgements
We thank the Ministry of Environment and Forests, Govt. of
India for financial support; Forest Departments at Campbell Bay
(Great Nicobar) for help at the field station; Andaman Public Works
Department for their logistic support; Dr. Dinkar Salunke, National
Institute of Immunology, New Delhi for the N-terminal sequencing
and MALDI-TOF mass spectrometry; Mr. A. Hafiz and Mr. M. Alam,
ICGEB New Delhi, for help in the parasite culture. Arindam Bhatta-
charyya is thankful to the University Grants Commission (UGC),
Govt. of India for Junior Research Fellowship.
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