Arachin derived peptides as selective angiotensin I-converting enzyme (ACE) inhibitors: Structure-activity relationship

Article abstract of DOI:10.1016/j.peptides.2010.02.022

Current attention focuses on mechanisms of controlling blood pressure through the inhibition of angiotensin I-converting enzyme (ACE). Bioactive antihypertensive peptides of food origin are increasingly gaining importance as alternates to synthetic drugs in hypertension therapy. The ACE inhibitory property of an enzymatic digest of arachin, the major storage globulin of peanut (Arachis hypogaea) has been demonstrated. The ACE inhibitory activity of a tripeptide (IEY) isolated from these digests has been characterized. Five synthetic structural analogs of this peptide (IEW, IKY, IKW, IEP and IKP) were assembled and their ACE inhibitory activity evaluated. Among these, the tripeptide IKP was a potent competitive inhibitor with an IC₅₀ of 7 ± 1 × 10^-6 M similar to that of the potent whey peptides IPP and VPP. The inhibition data of these peptide analogs have been rationalized through docking simulations using the tACE-lisinopril complex at 2 A? resolution (PDB: 1086). The best docking poses were located at the tACE catalytic site resembling the mode of inhibition exerted by lisinopril, the synthetic drug. The degree of inhibition by the peptides correlated with the coordination distance between the catalytic Zn(II) and the carbonyl oxygen of the peptide bond between the amino-terminal and middle residue. These studies illustrate that these peptides, like lisinopril, behave as transition state analog inhibitors and are useful in therapeutic intervention for blood pressure management.

Full text of DOI:10.1016/j.peptides.2010.02.022

Peptides 31 (2010) 1165–1176
Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
Arachin derived peptides as selective angiotensin I-converting enzyme (ACE)
inhibitors: Structure–activity relationship
V.K. Jimsheena, Lalitha R. Gowda∗
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore 570020, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Current attention focuses on mechanisms of controlling blood pressure through the inhibition of
angiotensin I-converting enzyme (ACE). Bioactive antihypertensive peptides of food origin are increas-
ingly gaining importance as alternates to synthetic drugs in hypertension therapy. The ACE inhibitory
property of an enzymatic digest of arachin, the major storage globulin of peanut (Arachis hypogaea)
has been demonstrated. The ACE inhibitory activity of a tripeptide (IEY) isolated from these digests has
been characterized. Five synthetic structural analogs of this peptide (IEW, IKY, IKW, IEP and IKP) were
assembled and their ACE inhibitory activity evaluated. Among these, the tripeptide IKP was a potent com-
petitive inhibitor with an IC50 of 7 1 × 10−6 M similar to that of the potent whey peptides IPP and VPP.
The inhibition data of these peptide analogs have been rationalized through docking simulations using
the tACE–lisinopril complex at 2 Å resolution (PDB: 1086). The best docking poses were located at the
tACE catalytic site resembling the mode of inhibition exerted by lisinopril, the synthetic drug. The degree
of inhibition by the peptides correlated with the coordination distance between the catalytic Zn(II) and
the carbonyl oxygen of the peptide bond between the amino-terminal and middle residue. These studies
illustrate that these peptides, like lisinopril, behave as transition state analog inhibitors and are useful in
therapeutic intervention for blood pressure management.
Received 12 January 2010
Received in revised form 25 February 2010
Accepted 25 February 2010
Available online 7 March 2010
Keywords:
Bioactive peptides
Hypertension
Tripeptide inhibitors
Molecular docking
Protease-P
Arachis hypogaea
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid), lisinopril
(N2-[(1S)-1-carboxy-3-phenylpropyl]-l-lysyl-l-proline)
and
Hypertension is a major public health problem, the preva-
lence of which is increasing alarmingly in developing as well
as developed countries. Being stealthy in the progress of the
disease, it can cause blood vessel changes in the retina, abnormal
thickening of the heart muscle, stroke, heart attack, and arterial
aneurysm, and it is a leading cause of chronic renal failure. Even a
moderate elevation of arterial blood pressure leads to shortened
life expectancy [14]. Renin–angiotensin system (RAS) plays a
vital role in controlling hypertension. Angiotensin converting
enzyme (ACE; peptidyl dipeptide hydrolase, EC 3.4.15.1), a type
I membrane anchored dipeptidyl carboxypeptidase, by virtue
of its ability to convert angiotensin I to a potent vasoconstric-
tor angiotensin II and by abolishing the vasodilator effects of
bradykinin by degradation, operates as a key enzyme in the RAS
[37]. A plethora of potent inhibitors have been discovered or
synthesized that mainly differ from one another in potency, route
of elimination, duration of action, being either prodrugs or active
drugs. Synthetic ACE inhibitors, captopril (2S)-1-[(2S)-2-methyl-
enalapril ((2S)-1-[(2S)-2-[(2S)-1-ethoxy-1-oxo-4-phenylbutan-
2-yl]aminopropanoyl]pyrrolidine-2-carboxylic acid), which are
developed based on snake venom peptide scaffold, have estab-
lished themselves in the therapy of hypertension, congestive
heart failure and diabetic neuropathy [6]. Albeit their established
effectiveness, the inherently associated adverse side effects such
as skin rashes, dry cough and angio-edema necessitate the need to
find more natural and safer alternatives [35].
Peptides and proteins play a physiological role beyond being
sources of metabolic energy and essential amino acids. The specific
sequences of bioactive peptides hidden or latent in a food protein
sequence can be released during proteolysis, in food processing
or by gastrointestinal digestion and thereby provide physiolog-
ical benefits after release. Varied bioactivities in a plethora of
hydrolyzed proteins introduce a completely new dimension to
be considered while enumerating and describing dietary protein
quality [36]. These peptides containing only a few amino acids
(di or tri peptides) will be able to cross the digestive epithelial
barrier and enter the blood stream, which allows them to reach
peripheral organs and exert effects. Bioactive peptides are increas-
ingly becoming important as starting points for drugs and drug
related compounds. Among the various bioactive peptides, the
∗
Corresponding author. Tel.: +91 821 2515331; fax: +91 821 2517233.
E-mail addresses: lrg@cftri.res.in, lrgowda@yahoo.com (L.R. Gowda).
0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2010.02.022

1166
V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
antihypertensive peptides have been studied extensively.
plethora of antihypertensive peptides from enzymatic
India. Amino acid standards (Pierce H.) and phenylisothiocynate
(PITC) were from Pierce Chemical Company, Rockford, Illinois,
USA. All other reagents and chemicals used were of analytical
grade.
A
hydrolyzates of various sources like milk [5], fish [17], fermented
milk [12], mushroom [16], soy bean [25,51], insect protein [47],
peanut flour [34] and egg [24] have been demonstrated to inhibit
ACE both in vivo and in vitro. Although there is vast information
on the production and characterization of antihypertensive pep-
tides, the information on in vitro structure–activity relationship
is sparse. Peptide Quantitative Structure–Activity Relationship
(QSAR) modeling has been used for predicting peptide struc-
tures with high ACE inhibitory activity. Using physicochemical
descriptors, Pripp et al. [33] emphasized that increased side chain
hydrophobicity at the carboxy-terminus and decreased side chain
length of the penultimate amino acid exemplified ACE inhibitory
potential of peptides up to six amino acids in length. ACE inhibitors
reported are diverse and derived from a varied array of plant
and animal origin proteins by different enzymes and hydrolysis
times, advocate that a variety of peptides with various amino acid
sequences are able to inhibit ACE [46]. This study was undertaken
to optimize and illustrate a more systematic understanding of the
relationship between the peptide structure and ACE inhibitory
potential. The fact that storage proteins of oil seed meals are
repositories of bioactive peptides, yet underutilized led us to carry
out this study on the storage proteins of peanut (Arachis hypogaea).
The major proteins of peanuts are the storage globulins, arachin
and conarachin I and II, and comprise nearly 75–80% of the total pro-
teins [32]. These proteins are easily extractable in aqueous solvents.
The low lysine to arginine ratio in these proteins has long been rec-
ognized as having an anti atherogenic effect [10]. The sequence of
these storage proteins are intrinsically laden with bioactive pep-
tides that need to be harnessed.
2.2. Preparation of porcine kidney and lung ACE
ACE was extracted from porcine lung and kidney acetone pow-
der prepared in the laboratory. One gram of acetone powder was
extracted with 10 mL of 0.05 M sodium borate buffer pH 8.2 con-
taining 0.3 M NaCl and 0.5% Triton X-100 at 4 ◦C for 16–18 h. The
extract was centrifuged at 14,100 × g for 60 min at 4 ◦C. The super-
natant was dialyzed against the same buffer without Triton X-100
(1 L × 3) for 24 h and stored at −20 ◦C until used. The specific activ-
ities were 0.003 and 0.002 units/mg for porcine lung and kidney
ACE, respectively.
2.3. Protein estimation
Protein concentration was determined by the method of Brad-
ford [3]. Bovine serum albumin was used as the standard.
2.4. In vitro colorimetric assay for ACE
ACE activity was assayed by monitoring the release of HA from
the substrate HHL as described [18]. The assay mixture contained
0.125 mL of 0.05 M sodium borate buffer pH 8.2 containing 0.3 M
NaCl, 0.05 mL of 5 mM HHL and 0.025 mL of ACE enzyme extract.
The reaction was arrested after incubation at 37 ◦C for 30 min by the
addition of 0.2 mL of 1 M HCl. After stopping the reaction, 0.4 mL
of pyridine was added followed by 0.2 mL of BSC and mixed by
inversion for 1 min and cooled on ice. The yellow color developed
was measured at 410 nm in a spectrophotometer (Shimadzu UV
1601). One unit of ACE activity is defined as the amount of enzyme,
which releases 1 ␮mol of HA per min at 37 ◦C and pH 8.2.
In the present investigation, the ACE inhibitory property of a
Protease-P hydrolyzate of arachin, the major storage protein of
peanut has been demonstrated. The hydrolyzate was further frac-
tionated and the major ACE inhibitory tripeptide segregated from a
suite of peptides and characterized. Synthetic analogs of this pep-
tide were used to understand the structural features that contribute
to ACE inhibition potency. Their interaction with the active site of
human tACE was evaluated by molecular docking to comprehend
the structure and specific conformations adopted by the peptide in
the microenvironment to explain the differences observed in the
degree of ACE inhibition.
2.5. In vitro assay of ACE inhibitor activity
Porcine lung or kidney ACE was preincubated (10 min) with
different concentrations of the peptide fractions and the resid-
ual activity determined as described above. The IC50 value is
defined as the concentration of the peptide required to decrease
the ACE activity by 50%. The percent inhibition curves were plot-
ted using a minimum of five independent determinations for each
inhibitor concentration and IC50 values computed from the semi
logarithmic plots. A linear regression analysis was performed using
Origin 4.1 to evaluate the mode of inhibition and to determine
the dissociation constant (Ki). Various concentrations of HHL were
incubated with a fixed concentration of either porcine lung ACE
in the presence or absence of ACE inhibitor peptides at 37 ◦C. The
HA released was determined by the colorimetric method. The ini-
tial reaction velocities were calculated from the HA released. The
type of inhibition was evaluated from Lineweaver–Burke plot [21]
and the Ki was calculated from a Dixon plot of the same data
[9].
2. Materials and methods
2.1. Materials
Peanut (A. hypogaea) variety TMV-2 was obtained from Uni-
versity of Agricultural Sciences, Gandhi Krishi Vignana Kendra
(GKVK) Bangalore, India. Porcine lung and kidney acetone pow-
ders were prepared in the laboratory. Protease-P-Amano-P was
from Amano Pharmaceuticals, Nagoya, Japan. Hippuryl-histidyl-
leucine (HHL), hippuric acid (HA), sodium tetraborate, Triton
X-100, coomassie brilliant blue G-250 (CBB G-250), coomassie
brilliant blue R-250 (CBB G-250), O-(benzotriazol-1-yl)-N,N,Nꢀ,Nꢀ-
tetramethyluronium hexa fluorophosphate (HBTU), triethylamine,
ethanedithiol (EDT) and trifluoroacetic acid (TFA) were obtained
from Sigma–Aldrich Chemicals Co., St. Louis, MO, USA. Fmoc (9-
fluorinylmethyloxycarbonyl) protected amino acids and resins
were from Novabiochem, Merck KGaA, Darmstadt, Germany
and Chem Impex International, Inc., USA. Methanol (HPLC
grade), acetonitrile (HPLC grade), diisopropyl ethyl amine (DIEA),
dimethyl formamide (DMF), dichloromethane (DCM), thioanisole,
1-hydroxybenzotriazole (HOBT), diethyl ether, pyridine and ben-
zene sulfonyl chloride (BSC) were from Spectrochem Pvt. Ltd.,
2.6. HPLC assay for the determination of ACE activity
The ACE activity was also verified by reverse phase high perfor-
mance liquid chromatography (RP-HPLC) assay reported [50]. The
product HA was separated from HHL by RP-HPLC on a Waters Sym-
metryshield octadecyl column (150 mm × 4.6 mm (i.d.), 5 ␮M) by
isocratic elution with 50% methanol containing 0.1% TFA at a flow
rate of 0.8 mL/min and detected at 228 nm.

V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
1167
2.7. Isolation of arachin
2.12. Fmoc solid phase peptides synthesis (Fmoc SPPS)
Arachin was isolated as described earlier [44]. A 10% (w/v)
slurry of defatted peanut flour in 10% NaCl (w/v) was extracted
by agitation for 4–6 h at 25 2 ◦C. The extract was clarified by
centrifugation 4300 × g for 45 min at 25 2 ◦C. Arachin was precip-
itated by the addition of solid (NH4)2SO4 to a final concentration of
40% (w/v). The precipitate obtained after centrifugation (6700 × g,
25 2 ◦C, 30 min) was redissolved in 10% NaCl. The (NH4)2SO4 pre-
cipitation was repeated three times to obtain pure arachin. The
precipitated arachin dissolved in water was dialyzed extensively
(1 L × 4) against distilled water, freeze dried and stored at 4 ◦C until
further use. The purity of protein was confirmed by SDS-PAGE,
amino-terminal sequencing by Edman degradation and amino acid
analysis.
The acid forms of the tripeptides were assembled on an auto-
matic peptide synthesizer (Model CS 136, CS Bio Co. San Carlos, CA,
USA) using standard Fmoc chemistry. Fmoc-L-Trp-2-chlorotrityl
and Fmoc-L-Pro-2-chlorotrityl resins preloaded with the first
amino acid were used for peptides containing carboxy-terminal
Trp and Pro respectively. Fmoc-Tyr (tBu)-Wang resin was used for
peptides containing carboxy-terminal Tyr.
The sequence of the tripeptides was verified by amino-terminal
sequencing and amino acid analysis. Semi-preparative HPLC was
carried out using Shimadzu HPLC equipped with a LC8A pump on a
C-18 Shimpak column (25 cm × 21.2 mm (i.d.), 10 ␮M) and a binary
gradient of 0.1% TFA and 70% acetonitrile in water containing 0.05%
TFA at a flow rate of 15 mL/min traversing from 0% to 100% B in
60 min. Data were collected via a SPD-M10AVP photodiode array
detector at 230 nm. Purity of all the peptides was >99% as deter-
mined by RP-HPLC.
2.8. SDS-PAGE
SDS-PAGE (12.5% T, 2.7% C) was performed according to the
method of Laemmli [20]. The gel was stained for protein with 0.1%
coomassie brilliant blue R-250. The molecular mass of the arachin
subunits were calculated using a calibration curve of log molecu-
lar weight versus relative mobility of standard molecular weight
markers.
2.13. In vitro stability of the peptides
In vitro stability against gastric proteases was assessed by incu-
bating a 0.1% (w/v) peptide solution in 0.01 N HCl with pepsin for
2 h at 37 ◦C, the reaction was stopped by boiling in a water bath for
10 min and neutralized, and residual ACE inhibitor activity deter-
mined. Stability was similarly assessed using pancreatin in 0.02 M
Tris–HCl, pH 8.2 at 37 ◦C for 2 h.
2.9. Preparation of arachin derived ACE inhibitor peptides
One gram of isolated arachin was suspended in 5 mL of 0.1 M
Tris–HCl buffer pH 8.2 and digested with Protease-P (Amano-P from
Aspergillus niger (2200 U/mg of protein) using an enzyme substrate
ratio of 0.5%, 1.0%, 2%, 4%, 6%, 8% and 10% (w/w) at 37 ◦C for 4 h. The
pH of the reaction mixture was maintained at 8.2 by the addition of
HCl. The reaction was terminated by the addition of 100% TCA to a
final concentration of 10% and cooled on ice. The hydrolyzate was
centrifuged at 14,100 × g for 30 min at 4 ◦C to remove undigested
protein. The supernatant containing a suite of peptides was used
as the source of ACE inhibitor peptides. This suite of peptides was
fractionated by RP-HPLC on a semi-preparative C-5 Phenomenex
column (25 cm × 21.2 mm (i.d.), 10 ␮M) using a binary gradient of
0.1% TFA and 70% acetonitrile in water containing 0.05% TFA at a
flow rate of 10 mL/min traversing from 0% to 100% B in 60 min.
The peptides were detected at 230 nm. The peptides were further
purified on the same reverse phase column using an ion pairing
gradient of (A) 50 mM ammonium acetate and (B) 50 mM ammo-
nium acetate/acetonitrile (50:50). The peptide fraction showing
highest ACE inhibition was subjected to RP-HPLC on a C18 col-
umn (4.6 mm × 150 mm (i.d.), 5 ␮M) using TFA/acetonitrile solvent
as described previously.
2.14. Molecular docking
The model for ACE used in this study was derived from the co-
ordinates of the structure labeled 1086 in the Brookhaven Protein
Data Bank, which represents the human tACE bound to lisinopril at
2 Å resolution [28]. The structure of the tripeptides was constructed
and optimized using the polypeptide builder function of the public
domain web server PepBuild which uses SCRWL to predict the
conformations of the side chains and optimizes using rotameric
libraries (www.imtech.res.in/bvs/pepbuild/method.html) [41].
Explicit hydrogens in the peptide structures were assigned using
PepBuild and saved in the MOL format. The possibility of bind-
ing, precise location of binding sites and mode of binding for
each ligand was carried out independently using an automated
docking software, Molgro Virtual Docker 2008, version 3.2.1
(Molgro ApS, Aarhus, Denmark, http://molegro.com), that is
based on guided differential evolution and a force field based
screening function [43]. Possible binding conformations and
orientations were analyzed by clustering methods, embedded in
MolDock.
All docking studies were carried out using the crystal struc-
ture of tACE complexed with the inhibitor lisinopril (PDB: 1086)
as the template. All water molecules and the inhibitor lisinopril
were excluded, whereas zinc and chloride atoms were retained in
the active site, fixed to their crystal positions, throughout the dock-
ing process. The molecular structures of the imported ligands were
manually checked before docking and corrected. The initial geom-
etry and topologies of the protein and the ligands were retrieved
to the docking software Molgro Virtual Docker. The binding site
was computed within a spacing of x: 37.26, y: 35.35 and z: 46.21
such that the binding site of tACE was well sampled with a grid
resolution of 0.3 Å. Each ligand (peptides) was then individually
docked into this grid using the MolDock Optimizer algorithm, and
its interactions monitored using detailed energy estimates. Cluster-
ing was performed based on the similarities in binding modes and
strengths in these cycles and ignoring similar poses. A value of pop-
ulation size and maximum interactions 100 and 10,000 respectively
were used for each run and 5 best poses were retained for each lig-
2.10. Sequence determination of the peptides by Edman
degradation
The amino-terminal sequence was determined by Edman degra-
dation using an automated Applied Biosystems 477-A gas phase
sequencer equipped with an online detection system for PTH-
amino acids. ␤-Lactoglobulin was used as standard to validate the
performance of the system.
2.11. Amino acid analysis
The purified peptides were hydrolyzed in vacuum at 110 ◦C in
6 N HCl for 24 h. Amino acid analysis was performed by pre-column
derivatization with PITC. The phenylthiocarbomoyl amino acids
were hydrolyzed by RP-HPLC [2].

1168
V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
Fig. 1. (A) SDS-PAGE profile (12.5% T, 2.7% C) of Protease-P hydrolyzates of arachin. Lane M. Molecular weight markers (phosphorylase b (Mr 97,400 Da); bovine serum
albumin (Mr 66,300 Da); ovalbumin (Mr 43,000 Da), carbonic anhydrase (Mr 29,000 Da), soybean trypsin inhibitor (Mr 20,100 Da), lysozyme (Mr 14,300 Da), lane 1: arachin
(control), lanes 2–7: arachin digested using enzyme to arachin ratio of 0.5, 1, 2, 4, 6 and 8%, respectively. ∼75 ␮g of protein loaded per well. B. The effect of enzyme/arachin
ratio on ACE inhibitory activity of the digests. ACE inhibition was assayed using the colorimetric method.
and. Lisinopril was docked into the template structure as a positive
control and the RMSD obtained. The docking mode obtained from
this validation exercise superposed well with that of the crystal
structure given credence by the minimal RMSD of 0.4 Å obtained.
Predicted low binder tripeptide, AVP [31] was used as negative
control. The software Discovery Studio Visualizer 2.5.1 (Accelrys
Software Inc., http://www.accelrys.com) was utilized to establish
hydrogen bonds and hydrophobic interactions between residues at
the ACE active site and the peptide poses.
3.2. ACE inhibition of arachin fungal protease digests
The arachin extracted from peanut meal was digested with
different concentrations of Protease-P (Amano-P from A. niger
(2200 U/mg of protein). The extent of digestion was analyzed by
SDS-PAGE (Fig. 1A). The results indicate that maximum digestion
was accomplished with 2% (w/w) of enzyme. A further increase
in the enzyme substrate ratio had appeared to have no effect on
the digestion pattern (Fig. 1A, lanes 3–6). The acidic subunits (A–C)
of molecular mass 35,500–40,500 Da were extremely susceptible
to proteolysis. Similarly the basic subunit (Fig. 1A) was degraded
completely. The digestion with different enzyme substrate ratios
results in the release of peptides of ≈14,300–20,000 Da that is resis-
tant to further digestion. The ACE inhibitory potential of the crude
proteolytic digests was evaluated against porcine lung ACE. The
results indicate that maximum inhibition (47 2%) was achieved
at an enzyme substrate ratio of 4% (w/w). Increasing the enzyme
concentration thereafter did not enhance the ACE inhibitory activ-
ity (Fig. 1B). These results are in concurrence with changes observed
in the peptide profile on increasing enzyme concentration (Fig. 1A).
The ACE inhibitory potential of the digests towards lung and kidney
was similar. The suite of peptides following Protease-P digestion
(4%, w/w) was selected for further characterization.
2.15. Data analysis
For all the measurements, a minimum of 3–5 replicates was
taken for data analysis. Using the software Origin 4.1, all of the
values were averaged and mean values are reported. The standard
deviation of 5 different replicate data was also tabulated.
3. Results
3.1. Isolation of arachin
Arachin, the major storage globulin of peanut was isolated to
apparent homogeneity. The subunit architecture was evaluated by
SDS-PAGE (Fig. 1A). The results show that it is contains 12 sub-
units as reported earlier [1]. The amino acid composition (data
not shown), amino-terminal sequence and estimated molecular
mass of the subunits (Fig. 1A and Table 1) correspond to that
reported for arachin in the Protein Data Bank. The subunits A, B
and C (Fig. 1A) based on their molecular mass and amino-terminal
sequence (Table 1) can be classified as the acidic subunits. The
release of glycine as the amino-terminal of the subunit D and
its mass renders it as a basic subunit. These results indicate the
purified arachin is homogenous and can be used for further analy-
ses.
3.3. Isolation of ACE inhibitor peptide
Semi-preparative RP-HPLC of the Protease-P hydrolyzate on a
C-5 column resulted in three major peptide fractions with a consid-
erable number of minor peptides (Fig. 2). Among the major peptide
fractions, fraction 1 (retention time = 30.74 min) exhibited the high-
est ACE inhibitor activity (Fig. 2 inset). This peptide fraction was
subsequently fractionated by ion-pair chromatography using an
ammonium acetate/acetonitrile solvent system. This peptide frac-
tion was further re-chromatographed on the same column using
the TFA/CH3CN solvent system. The RP-HPLC profile showed a sin-
Table 1
The amino-terminal sequences of arachin subunits.
Subunit no.a
Amino-terminal sequence
Mr (kDa)
PDB accession no.
A
B
C
D
ISFYQQPEENACQQQYLNAQQ. . .
ISFYQHPEENAAQFQYLNA. . .
VTFRQGGEENECQFQRLNA. . .
42
38
35
24
1
1
1
1
ACH91862
ABL14270
ABI17154
AAW56067
GIEEGIRGASVKSNDGYNEQADIYXPQ. . .
a
The subunit numbers as labeled in Fig. 1A. Following SDS-PAGE the protein bands were transferred to a PVDF membrane in 0.01 M CAPS-10% methanol buffer (pH 11)
and stained with coomassie brilliant blue R-250. The bands were excised, washed with methanol and loaded directly to the Applied Biosystems 477A automated gas phase
sequencer for N-terminal sequencing by Edman degradation.

V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
1169
Fig. 2. RP-HPLC profile of Protease-P digest of arachin at an enzyme/arachin
ratio of 4% (w/w). The peptides were resolved on a semi-preparative C5 column
(25 cm × 21.2 mm (i.d.), 10 ␮M) using a gradient of acetonitrile and water contain-
ing 0.05% TFA. Inset: ACE inhibitory activity of peptide fractions, assayed by the
colorimetric method using porcine lung ACE and HHL as the substrate.
gle peak indicating the peptide was homogeneous. The purified
peptide inhibited both porcine lung and kidney ACE.
3.4. Characterization of the ACE inhibitor peptide
The amino acid composition of the purified peptide was deter-
mined after hydrolysis in vacuo with 6 M HCl at 110 ◦C for 24 h. The
amino acid composition indicated the presence of only 3 amino
acids, Ile, Glu and Tyr in equimolar concentration. The amino acid
sequence of the peptide was found to be Ile–Glu–Tyr (IEY) and theo-
retical molecular weight of the peptide was ≈423 Da. The sequence
IEY corresponded to the sequence Ile5–Tyr7 of the 21,000 Da sub
unit of arachin (PDB: P20780).
The tripeptide IEY was assembled by solid phase peptide synthe-
sis using Wang resin and Fmoc chemistry and purified by RP-HPLC.
The homogeneity of the peptide was validated by the single peak
observed with a retention time of 18.97 min (Fig. 3A). The purity
was >99%. The peptide sequence was verified by Edman degra-
dation. The inhibitory potential of the synthetic peptide towards
porcine lung ACE was evaluated. The synthetic peptide was an
inhibitor of porcine lung ACE with an IC50 of 182 2 ␮M (Table 2
and Fig. 4A). The effect of the tripeptide on varying substrate con-
centrations plotted as a Lineweaver–Burk plot (data not shown)
indicated that the synthetic peptide was a competitive inhibitor
with respect to the substrate HHL. The equilibrium dissociation
Fig. 3. RP-HPLC profile indicating the homogeneity of the peptides synthesized
by Fmoc chemistry. The purity of the peptides was analyzed using a C18 column
(150 mm × 4.6 mm (i.d.), 5 ␮M) on a Waters Associate HPLC, with a gradient of 0.1%
TFA and 70% acetonitrile containing 0.05% TFA. (A) IEP, IEY and IEW. (B) IKP, IKY,
IKW and IK*P.
constant Ki calculated from the Dixon plot of the data shown in
Fig. 4B was 83.53 ␮M (Fig. 4C). The IC50 value of IEY is several fold
higher than that reported for the antihypertensive tripeptides VPP
(9 ␮M) and IPP (5 ␮M) derived from milk protein [31].
3.5. Peptide analogs of IEY and ACE inhibition
Previous structure–activity relationship studies predict that a
tripeptidewith abulky aliphaticresidueattheamino-terminal such
as Leu/Ile, a positively charged residue at the middle position and
an aromatic residue or Pro at the carboxy-terminus will emerge as
potent ACE inhibitor peptides [49]. The characterized peptide IEY
satisfies these criteria with the exception that the middle position
holds a negatively charged acidic amino acid. Could this difference
be responsible for the elevated IC50 of IEY? To answer this question,
tripeptide analogs of IEY using various amino acid substitutions at
the middle and carboxy-terminal positions were synthesized and
the IC50 values for the inhibition of porcine lung ACE determined.
Initially, the carboxy-terminal Tyr was replaced with a bulkier aro-
matic residue Trp (W) or Pro (P). For a second replacement, the
negatively charged middle amino acid Glu (E) was exchanged for a
positively charged Lys (K). Consequently, the following peptides
IEW, IEP, IKY, IKW and IKP were synthesized. These tripeptides
were assembled by standard Fmoc chemistry using Fmoc protected
amino acids as building blocks and were purified by preparative RP-
HPLC. The single peaks obtained by analytical RP-HPLC thereafter
suggest that the peptides were pure and homogenous (Fig. 3A and
B). The purity of all the peptides was >99%. The sequences of the
synthetic peptides were further validated by Edman degradation
and amino acid analyses.
Table 2
Inhibition of porcine lung ACE activity by the tripeptides
calculated as IC50 (␮M).
Peptide
IC50 (␮M)
IEY
IEW
IEP
IKY
IKW
IKP
IEP + IEY
IEP + IEW
IEY + IEW
IEP + IEY + IEW
182
104
18
34
19
4
3
2
3
2
1
5
4
4
3
7
132
101
107
146
The values are an average of five replicates. The IC50 val-
ues were determined using the colorimetric assay and
validated using the HPLC method.

1170
V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
Porcine lung ACE was preincubated for 10 min with varying con-
centrations of each of these peptides. The residual ACE activity
was determined as described. Semi logarithmic plots were used to
determine IC50 values (Table 2). The results suggest that substitut-
ing Pro for Tyr at the carboxy-terminal of the tripeptide increased
the potency 10-fold whereas the bulkier Trp did not significantly
enhance inhibition (IC50 182–104 ␮M). Replacing the acidic Glu
with Lys increased the inhibitory potential by 5-fold. The synergis-
tic effect of these two changes was evident from the much lower
IC50 of IKP which was 17-fold more potent than the isolated peptide
IEY. The IC50 of 7 1 ␮M of IKP is similar to that reported for potent
antihypertensive peptides IPP and VPP derived from milk proteins.
The observation that the parent crude digest, which contains a suite
of peptides, always exhibits a much higher inhibition than the pep-
tides in isolation led us to evaluate synergism if any, between the
peptides. The results (Table 2) however suggested that there was
no apparent increase in inhibitory potency among these peptides
used in combination. These results coupled with the competitive
inhibition observed for the individual peptides are suggestive of
a single binding site on ACE. Kinetic analysis of the most potent
tripeptide IKP-porcine lung ACE complexation was consistent with
a reversible and competitive mechanism of inhibition (results not
shown) with respect to the substrate HHL. The binding affinity (Ki)
of IKP to ACE was 2.7 × 10−6 M as calculated from the Dixon plot.
3.6. In vitro stability of the peptides to gastrointestinal digestion
ACE inhibitor peptides for therapeutic use must be resistant to
gastrointestinal proteolysis once they are ingested orally in order to
reach the target tissue and exert an antihypertensive effect. The sta-
bility of each of the purified peptide was assessed in vitro in order
to predict their antihypertensive potential in vivo. The inhibitory
potential of all the peptides were preserved after digestion with
pepsin. However, the tripeptides IKY and IKP were partially sta-
ble to the consecutive digestion with pancreatin (contains trypsin
and chymotrypsin) (Table 3). This decrease in ACE inhibition can be
attributed to the hydrolysis by trypsin at the carboxyl-end of Lys
of the peptides. Among the synthetic peptides, IKP was the most
potent inhibitor of porcine lung ACE, yet it was more susceptible to
in vitro digestion with pancreatin (Table 3). Therefore to increase
the in vitro stability and to decrease the susceptibility to pancreatin
digestion, the tripeptide IK*P was synthesized replacing l-Lys with
Table 3
In vitro stability of the peptides to gastrointestinal proteasesa.
Peptide
IEP
Protease
ACE inhibition (%)
Control
Pepsin
100
99
Pancreatin
100
IEY
IEW
IKP
IKY
Control
Pepsin
Pancreatin
100
99
100
Control
Pepsin
Pancreatin
100
100
100
Control
Pepsin
Pancreatin
100
100
66
Fig. 4. Kinetic analysis of the tripeptide IEY/porcine lung ACE complexation con-
sistent with a reversible and competitive inhibition. ACE activity was determined
as described under methods either in the absence or presence of the indicated
tripeptide concentrations. (A) The semi logarithmic plot to determine IC50 of IEY,
(B) Michaelis–Menten curve showing the inhibition in presence of different con-
centration of IEY and (C) Dixon plot of the data in part B used to determine Ki (the
data represent means of triplicate experiments with SEM never exceeding 4%).
Control
Pepsin
Pancreatin
100
99
75
IKW
Control
Pepsin
Pancreatin
100
100
97
a
The ACE inhibition is the mean of three experiments. The SEM was ≤3% in each
case.

V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
1171
Table 4
Experimental binding constants, predicted binding energies and Zn(II) coordination distances of tripeptide with tACE (PDB: 1086).
Ligand
Ki (␮M)
Affinity energy (kJ/mol, pose)
Zn(II) coordination
Distance (Å)
Atom
Lisinopril
IKP
IEP
IKW
IKY
IEW
IEY
0.001
2.66
7.24
2.141
2.730
3.036
3.299
3.445
–
Carboxyl group of lisinopril
−13.45 (00)
−15.43 (02)
−18.20 (00)
−15.80 (00)
−17.50 (01)
−15.57 (00)
Carbonyl oxygen of the peptide bond between I and K
Carbonyl oxygen of the peptide bond between I and E
Carbonyl oxygen of the peptide bond between I and K
Carbonyl oxygen of the peptide bond between I and K
No Zn(II) coordination
8.5
12.96
55.01
83.53
–
No Zn(II) coordination
d-Lys. The peptide albeit resistant to proteolysis by pancreatin did
not inhibit either porcine lung or kidney ACE at concentrations as
high as 5 mM. These results indicate that the stereo-specificity of
the amino acid residue plays a vital role in ACE inhibition.
with the corresponding crystal structure of tACE-lisinopril com-
plex (PDB: 1086). The best returned pose in presence of Zn(II) with
a docking score of 154.43 (Table 4), revealed the same ACE residues
involved in Zn(II) coordination. The main interactions considered
responsible for the high potency of this drug have been repro-
duced by this docking procedure (Tables 4–6). These include (1)
carboxy alkyl carboxylate and Zn(II) coordination and the H-bond
with Glu384 (OE2 atom) and (2) the H-bond between the carboxy-
terminal carboxylate of lisinopril interaction with Lys511 and Tyr520
at the S1ꢀ subsite. These results validate the docking accuracy for
further studies with the inhibitor tripeptides.
The best poses of the docking study of the tripeptides at tACE
catalytic site in presence of Zn(II) are shown in Fig. 5. The best
poses for each of these tripeptides was stabilized by H-bonds and
hydrophobic interactions with tACE residues (Tables 5 and 6). The
molecular docking of the tripeptides on the binding site of tACE
reveal that these peptides are buried deep inside the active site
channel similar to lisinopril in the tACE-lisinopril complex [28].
The molecular docking studies revealed that these six bioactive
peptides occupy mainly the S2ꢀ subsite and are also accommo-
dated in the hydrophobic pocket with hydrophobic interactions
with Phe512, Phe527, Phe457or Val518. The carbonyl oxygen of the
peptide bond between the P1 and P1ꢀ residues (Schechter and
3.7. Molecular docking
Automated docking simulations were performed with tACE and
the tripeptide inhibitors to correlate the amino acid substitutions
in the tripeptides with the changes in the ACE inhibitor potential
observed. Human sACE is composed of two homologous domains
(N and C-domain) having ≈60% sequence identity, each one with
a functional catalytic site that comprises the highly conserved
HEXXH Zinc binding motif [7,8,45], with C-domain being the dom-
inant angiotensin converting site [28]. As the sequence of tACE and
C-domain of sACE are identical; all the docking studies were carried
out with tACE.
A molecular docking algorithm Molegro Dock based on a heuris-
tic search algorithm that uses a cavity prediction algorithm [43] was
used to elucidate the mode of interactions between the tACE and
the tripeptide ACE inhibitors. To evaluate the effectiveness of the
MolDock programme in the docking of the inhibitor tripeptides,
we first docked lisinopril and compared the resulting geometry
Table 5
Residues of tACE having at least one atom at a distance of 3.5 Å around the docked peptide. The residues around crystallized lisinopril in the tACE (PDB: 1086) are also shown.
The residue numbers areas of tACE (PDB: 1086).
No.
tACE residues
Lisinopril
√
IEY
√
IEW
√
IEP
√
IKY
√
IKW
√
IKP
√
1.
2.
3.
4.
5.
6.
7.
8.
ALA354
ALA356
ARG522
ASP377
ASP415
CYS352
GLN281
GLU162
GLU376
GLU384
GLU411
HIS353
HIS383
HIS387
HIS513
LYS368
LYS511
PHE457
PHE512
PHE527
SER355
TRP357
TYR520
TYR523
VAL380
VAL518
GLN369
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
9.
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
Total
12
16
19
14
15
15
17
√
(
) indicates the presence of the residue.

1172
V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
Berger nomenclature) [38] of all the peptides except IEY and IEW
are positioned to coordinate the active site Zn(II) atom (Fig. 5). This
coordination coupled with that of His383, His387of the ␣13 helix
and Glu411 affords a tetrahedral geometry in semblance to the dis-
torted tetrahedral geometry of lisinopril/captopril-tACE complex.
The direct coordination with the catalytic Zn(II) and the carbonyl
oxygen of the peptide bond between Ile and Lys in the IKP is almost
the same order reported for captopril (2.32 Å). It is conceivable
that the distance between the peptide bond carbonyl oxygen and
Zn(II) account for the varied degrees of inhibition by these peptides.
Shorter the Zn(II) distance to the carbonyl oxygen of the peptide,
the greater the degree of inhibition (Table 4). This Zn(II) coordina-
Fig. 5. Predicted binding mode of peptide inhibitors to tACE (PDB: 1086). The best ranked docking pose is shown. Stereo image of catalytic binding site of tACE. Lisinopril
from the PDB structure was eliminated and peptides docked at the active site. Peptide ligands are shown in stick model. Zn(II) is shown as a circle. Dotted lines indicate
hydrogen bonds and bold line indicates Zn(II) coordination with the peptide backbone.

V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
1173
Table 6
Hydrogen bonds observed between the docked top ranked pose of peptides and tACE. The residue numbers are as of tACE (PDB: 1086).
tACE residues in H-bonding
Number of H-bonds and their corresponding distance (Å)
Lisinoprila
IEY
IKP
IEW
IEP
IKY
–
IKW
ALA354:O
1 (2.9)
–
–
–
1 (2.2)
2 (2.9, 2.9)
–
–
–
–
–
GLN281:NE2
GLU162:OE1
GLU162:OE2
HIS353:NE2
HIS513:NE2
LYS511:NZ
TYR520:OH
TYR523:OH
GLU384:OE2
ASP377:OD2
1 (2.6)
1(3.2)
–
–
–
1 (1.9)
–
–
–
1 (3.1)
1 (2.8)
–
–
–
–
–
–
–
2(1.6, 1.7)
–
3(2.3, 1.9,2.4)
1(2.3)
1(1.9)
1 (3.4)
1 (2.8)
1 (3.1)
1 (2.9)
1 (2.6)
1 (2.8)
1 (2.7)
–
1(1.9)
2(2.1, 1.7)
2(2.4, 2.4)
1(3.0)
2(2.71.6)
1(3.0)
–
1 (2.2)
–
–
–
1 (1.8)
1 (2.4)
1 (2.2)
–
1 (2.6)
2 (2.6, 1.8)
–
–
–
–
–
–
–
–
–
2(2.6, 1.6)
1(2.9)
–
–
–
2 (2.3, 2.2)
1 (1.9)
–
Total
8
4
6
1
6
10
10
–: H-bond not observed.
a
H-bonds observed in the crystallized tACE-lisinopril complex (PDB: 1086).
the substrate substance-P [26]. The functional importance of this
carboxy-terminal carboxylate docking interaction in ACE inhibition
is revealed by comparing IEY and IEW with the other tripeptides.
The absence of H-bond interaction of IEY and IEW with both Tyr520
and Lys511 of tACE (Fig. 5 and Table 6) are commensurate with the
elevated IC50 values. It is possible that by virtue of steric hindrance,
the aromatic residues of IEY and IEW prevent the H-bond formation
between the carboxy-terminal carboxylate and Lys511. Substitut-
ing Glu with Lys restores a hydrogen bond interaction between
the carboxy-terminal carboxylate of IKW with Lys511 and Tyr520
(Table 6). This also explains why IKW (IC50 = 19 2 ␮M) exhibits
a better ACE inhibition than IKY (IC50 = 34 3 ␮M), in addition to
the Zn(II) coordinating distances. The tripeptide IEP although lack-
ing a H-bond to Lys511 of tACE, a strong interaction observed with
Tyr520 is probably a significant part of the inhibitor registration
strategy. The most potent porcine lung ACE inhibitor among the
tripeptides is IKP (Table 2). IKP not only registers both the cru-
cial H-bond interactions with tACE (Lys511 and Tyr520) necessary
for carboxylate docking but also reveals an interaction with Glu162
(OE1) and Ala354 (O). The lysyl amine forms a very strong hydro-
gen bond with Glu 162 (OE1 atom, 1.9 Å) at the S1ꢀ subsite of tACE
probably responsible for the tight binding (2.7 × 10−6 M). The weak
H-bond between Glu162 (OE2, 3.4) observed with lisinopril is absent
with IKP similar to that observed for captopril and enalapril and
the bioactive whey peptides IPP and VPP [31]. The positioning of
H-bond from Tyr523 present in lisinopril-tACE complex is lost in
the IKP-tACE model. In contrast, the H-bond to the carboxyl group
of Ala 354 (O) between P1 and P1ꢀ is much stronger (2.2 Å). The two
H-bonds from His513 and His353 are also lost. The amino-terminal
Ile interacts with Phe512 and Val518 of the hydrophobic pocket.
The lisinopril-tACE and IKP-tACE complexes superimposed (Fig. 6)
suggest a similar inhibition mechanism.
tion distance for IKP is 2.73 Å and IKP is 3-fold more potent than
IEP for which the coordinating distance is 3.036 Å (Table 4).
The results show that bulky aromatic residues at the carboxy-
terminus (P2ꢀ) result in the carbonyl oxygen of the P1–P1ꢀ bond
being rotated such that it does not coordinate to Zn(II) (Fig. 5,
IEY and IEW) thus explaining the higher IC50 values (Table 2).
The replacement with a Lys (positive charge) for the Glu (nega-
tive charge) leads to a weak Zn(II) coordination and a concomitant
increase in the ACE inhibition (Table 2). These results are in accor-
dance with the prediction that a positively charged amino acid
residue adjacent to an aromatic residue is essential for ACE inhibitor
activity [19].
The H-bonding interactions between the carboxy-terminal car-
boxylate of lisinopril and Tyr520 and Lys511 of tACE provide a
solitary and significant mode of inhibitor registration strategy like
These findings highlight the importance of both carboxylate
docking and carboxy-terminal residue side chain interactions and
the coordination distance to the carboxyl oxygen in between P1
and P1ꢀ site, in defining peptide ACE inhibitor potency.
4. Discussion
Natural components in foods with prophylactic and therapeutic
benefits include bioactive peptides that exhibit antihypertensive
properties. ACE inhibitor peptides, as a part of food product or as a
nutraceutical, especially when derived from a food protein of plant
or animal origin, are of functional interest as they would have no
harmful side effects [15]. A very large cache of food derived antihy-
pertensive peptides have been categorized based on their origin:
animal derived, plant derived and microorganisms [15]. Despite
Fig. 6. The ball and stick models of IKP (cyan) and lisinopril (red) superimposed at
the active site of tACE (PDB: 1086). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of the article.)

1174
V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
this vast array of bioactive peptides as an alternate line of therapy to
control mild hypertension, the exact mode and mechanism of ACE
inhibition is not clearly understood other than reported in vitro and
in vivo studies. The available crystal structures of human sACE and
tACE [8,26–28] with deep insights to the specificity and physiolog-
ical significance of the active site are a platform for understanding
the mode of inhibition of food derived peptides.
three z-scores of amino acids and proposed ACE inhibitor pep-
tides with higher potency than reported earlier. For tripeptides, the
most preferred residue for the amino-terminus was a hydrophobic
amino acid, a positively charged middle amino acid and favored
an aromatic residue at the carboxy-terminus. Accordingly, the ACE
inhibitory tripeptide of arachin ‘IEY’ fulfills these criteria with
respect to the amino- and carboxy-terminus except for the middle
residueGlu which is negativelycharged. Giventhese considerations
we questioned whether substituting Lys (positively charged) would
enhance the ACE inhibition efficiency. The peptide IKY was assem-
bled by chemical synthesis and the IC50 (34 3 ␮M) for porcine
lung ACE, showed it was six times more potent (Table 2). Replac-
ing Tyr with a bulkier Trp (IKW) increased the potency two times
(Table 2). Kobayashi et al. [19] showed that IKW was four times
more potent than IKY against rabbit lung ACE. Therefore among
the aromatic residues, the largest amino acid Trp at the carboxy-
terminus showed highest ACE inhibitory activity. A substitution of
Lys for Arg in tripeptide reportedly had little effect on ACE inhibi-
tion [19]. In this study, the Arg replacement was not studied. Wu
et al. [49] also show that a tripeptide IKY had a log IC50 of −0.68.
Our results are in accordance with the prediction that a positively
charged residue adjacent to an aromatic residue is essential for a
high ACE inhibitory activity. Different ACE inhibition assays, source
of ACE and calculations for the median inhibitory concentration
(IC50) hamper the comparison of ACE inhibitory activities of these
tripeptides to that reported in literature.
In this study, IKP was the most potent ACE inhibitory peptide
with an IC50 value comparable to VPP and IPP (5 and 9 ␮M), the
famous antihypertensive milk peptides [11,23]. The tripeptide IKP
is also located within the glycinin GI precursor of soybean and
legumin I precursor of garden pea [49] and the observed log IC50
was 0.44 as against the QSAR predicted 0.37. Although the most
favored carboxy-terminus amino acids reported were aromatic
amino acids, our study shows that Pro was superior, as IKP was
three times more potent than IKW (Table 2). These results coupled
with the extremely high potency of the well known milk tripep-
tides IPP and VPP, exemplify Pro as the favored carboxy-terminus
amino acid. Among the N-carboxy methyl transition state peptide
analogs, those with Lys-Pro at the carboxy-terminus inhibited ACE
at the nanomolar levels [30]. Cheung et al. [4] also demonstrated
that the inhibitory potency of dipeptides with Trp, Tyr or Pro at
the carboxy-terminus were 300-fold higher than Gly. In summary,
a carboxy-terminal Lys-Pro is an important facet of ACE peptide
inhibitors.
The health promoting properties of peanut protein have been
well documented. Arachin and conarachin, the major storage glob-
ulins of peanut account for nearly 75% of the total protein [32].
Arachin and its molecular species, separated and identified by
amino-terminal analyses (Fig. 1A), were similar to that reported
earlier [1,39–40]. The high arginine to lysine ratio of arachin
coupled with the putative antihypertensive peptide sequences
encrypted in the available protein sequence data for arachin sub-
units was the premise to prepare enzymatic hydrolyzate and study
their antihypertensive potential. Accordingly, the proteolysis of
arachin was engineered and Protease-P hydrolyzate contained
a suite of potent ACE inhibitory peptides. The SDS-PAGE pro-
file shows the disintegration of the arachin larger subunits on
hydrolysis, leaving no visible bands of the high molecular weight
bands (Fig. 1A). It could therefore be said that it is the small
peptide units that are responsible for ACE inhibition. Both the
acidic and basic subunits of arachin were vulnerable to diges-
tion, similar to that observed for roasted peanuts [34]. An array of
proteases viz pepsin, trypsin, chymotrypsin, pronase and actinase
have been used to generate hydrolyzates that exhibit ACE inhibi-
tion. Previously Mallikarjun Gouda et al. [25] demonstrated that
among several proteases used to hydrolyze glycinin, the soybean
storage protein, Protease-P digests showed the highest ACE inhi-
bition. Very recently Quist et al. [34], in an in vitro study, used
pepsin–pancreatin to simulate human digestion and alcalase an
industrially important enzyme to digest peanut flour. The alcalase
system produced more potent antihypertensive peptides com-
pared to the pepsin–pancreatin system.
The peptides in the enzyme digests were subsequently fraction-
ated and purified (Fig. 2). ACE inhibitory activity could be ascribed
to a tripeptide IEY. The sequence corresponded to the amino acids
5–7 of arachin segment (PDB: P20780) and in close agreement
to the determined amino acid composition. This peptide is a true
inhibitor and therefore useful for therapeutic intervention in blood
pressure management. Previously, we isolated a penta-peptide
from the enzymatic hydrolyzates of glycinin, the major storage
protein of soybean [25]. Very recently, a tetrapeptide (KFAR) iso-
lated from an alcalase treated peanut hydrolyzate showed an IC50
of 134.4 ␮g/mL [13], which is much higher in comparison to the
tripeptides (Table 2). Liu et al. [22] used antibodies raised against
an ACE inhibitory peptide isolated from lactic acid bacteria fermen-
tation of soybean meal extracts to detect ACE inhibitor peptides.
Several other plant proteins are a source of various peptides with
antihypertensive activity [15]. The chemically synthesized tripep-
tide (IEY) was a competitive inhibitor (Fig. 4C) with an IC50 of
≈182 4 ␮M. This IC50 value is very high when compared to the
ACE inhibitory peptides reported for other food proteins [15].
The ACE inhibitor peptide derived from arachin contains Ile at
the amino-terminus and Tyr, at the carboxy-terminus. Cheung et
al. [4] suggest that the most favored amino-terminal residues are
branched chain amino acids and Pro is among the most favored
carboxy-terminus residues. Pripp et al. [33] thoroughly examined
ACE inhibitory peptides by QSAR methodology using physico-
chemical descriptors. Their results emphasized that increased side
chain hydrophobicity at the carboxy-terminus and decreased side
chain size of the penultimate amino acid enhanced ACE inhibitory
potential for peptides containing up to six amino acids and no
such relationship existed for the amino-terminal residue. Further,
Wu et al. [49] modeled food based di and tripeptides from the
The physiological antihypertensive effect of ACE inhibitory pep-
tides is guaranteed by their bio-availability and predominantly
characterized by their resistance to gastrointestinal degradation
and efficient intestinal absorption [48]. In vitro simulated gastric
digestion provides practicalmimics ofthe fate ofsuch peptides after
oral administration. Although IKP showed the highest inhibitory
activity, it was relatively less stable under gastrointestinal con-
ditions compared to the other peptides of this study (Table 3). A
D-amino acid substitution although increased the stability yet lost
its ACE inhibitory property. The tripeptides IKP, IKW and IKY exhibit
promising properties for ACE inhibition in vitro plausibly due to
their small size and hydrophobic character. Small peptides can
enter the peripheral blood stream intact due to their low molecular
size and exert systemic effects or produce local effects in the diges-
tive tract [53]. Further it has been suggested that ACE inhibitory
peptides may exert an additional antihypertensive effect by inhibit-
ing chymase [52].
There exists very limited information on the relationship
between primary structure and ACE inhibitor potency of food
protein derived antihypertensive peptides.
A more profound
understanding of the tripeptide inhibitor binding mode to ACE has
many potentially favorable consequences. Therefore using molec-

V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
1175
ular docking studies, our aim was to understand the exact mode of
interaction of these tripeptides with human tACE catalytic site and
compare them with the manner in which captopril and lisinopril,
the synthetic drugs complex with ACE and inhibit it. As a conse-
quence of human tACE and C-domain of sACE being identical the
molecular modeling was carried out using the tACE-lisinopril com-
plex X-ray crystal structure [28]. The modeling results with tACE
(Fig. 5) indicate that the tripeptides bind to ACE with high similar-
ity to lisinopril. The best poses obtained for the tripeptides IKP,
IKY, IKW and IEP show the coordination of the carbonyl group
between P1 and P1ꢀ residues, directly with the catalytic Zn(II) at
the active site of tACE. Coupled with this, Zn(II) is coordinated
to His383, His387 and Glu411 in a distorted tetrahedral geome-
try, inhibiting ACE. The ACE inhibitor potency follows the order
IKP > IEP > IKW > IKY > IEW > IEY. The absence of Zn(II) coordination
in the tACE complex with IEW and IEY explains their very high IC50
values. As the distance between the coordinating carbonyl oxygen
of the peptide and Zn(II) increases, ACE inhibition decreases. IPP and
VPP, the milk peptides coordinate to Zn(II) through the carbonyl
oxygen at 2.51 and 2.66 Å respectively and IPP is the more potent
ACE inhibitor [31]. Similarly IKP (2.71 Å) with the shortest coordi-
nation distance, is the most potent inhibitor among the tripeptides
tested (Table 2). Interestingly, the trend of relative inhibitor poten-
cies correlates with the coordinating distance between Zn(II) and
the peptides (Table 4). A tryptophan analog at the P2ꢀ position of
lisinopril resulted in a 25–100-fold higher C-domain selectivity as
compared to lisinopril [29]. The direct interaction with Zn(II) in
the potent synthetic drugs is through the thiol group of capto-
pril and the carboxyl groups of enalaprilat and lisinopril [27] and
not the peptide bond, and are much shorter (2.1 Å for lisinopril),
which probably explains the IC50 in the nanomolar range. In the
Drosophila homologue of ACE which has 40% sequence identity,
the catalytic Zn(II) binds to the carbonyl group between P1 and P1ꢀ
site [42]. The distance between the phenyl carbonyl of the analog
and the Zn(II) in the C-domain was 2.73 Å [29].
effort of identifying a tripeptide from enzymatic digestion of
arachin, the storage protein of peanut (A. hypogaea) and demon-
strating its ACE inhibiting capacity has led to the design and
synthesis of potent tripeptide ACE inhibitors. The tripeptides have
favorable competitive type inhibitory properties and are resis-
tant to rapid biodegradation by proteases of the gastrointestinal
tract. The molecular modeling technique coupled with the biolog-
ical assays for ACE inhibition indicates that the tripeptides bind
at the catalytic cleft of tACE with the carbonyl carbon of P1–P1ꢀ
residue coordinating Zn(II) to complete the tetrahedral geometry.
The interaction of the tripeptide IKP with tACE, which has pro-
nounced in vitro ACE inhibitory activity (IC50 = 7 ␮M) is similar to
lisinopril and can be considered as a transition state inhibitor and
therefore find applications in therapeutic blood pressure manage-
ment.
Acknowledgements
We thank Dr. V. Prakash, Director, CFTRI, Mysore and Dr. A.G.
Appu Rao, Head, PCT, CFTRI, Mysore for their advice and use-
ful discussions. VKJ acknowledges a Senior Research Fellowship
from CSIR, India. This work was carried out under CSIR-Networking
Project CMM-0014 and MLP-077.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.peptides.2010.02.022.
References
[1] Bhushan R, Agarwal R. Reversed-phase high-performance liquid chromato-
graphic, gel electrophoretic and size exclusion chromatographic studies of
subunit structure of arachin and its molecular species. Biomed Chromatogr
2006;20:561–8.
The relative inhibitor potencies of the synthetic drugs were
attributed to the number of interactions in the crystal complex with
tACE. No such trend was observed with the tripeptides (Table 5).
IEW, which is ≈15 times less efficient than IKP shows the highest
number of interactions with tACE. The observation that both IEW
and IEY lack the H-bonds to Lys511 and Tyr520 whereas IKW and IKY
form these bonds suggests that the Lys at the P1 position orients the
peptide favorably for this H-bond formation with the carboxylate of
the P2ꢀ carboxy-terminus. In the structure of tACE bound to lisino-
pril, the terminal P2ꢀ is stabilized by Lys511 and Tyr520 [42]. The best
poses obtained for IKP reveal interactions with ACE via H-bonding
similar to the inhibition mode of the common drug lisinopril (Fig. 6,
Tables 5 and 6). In addition, the atoms coordinating the catalytic
Zn(II) resemble the tetrahedral coordination geometry of lisinopril.
It is conceivable that the tripeptide IKP like lisinopril is a transi-
tion state analog and inhibits ACE through a mechanism similar
to that of the drug. The molecular docking studies indicate that the
Zn(II) coordination distance between the P1 and P1ꢀ carbonyls in the
tripeptides is important for ACE inhibition potency. This molecular
docking study has paved the way to explain the biological activi-
ties of tripeptide ACE inhibitors and provides a platform to design
better peptide inhibitors.
[2] Bidlingmeyer BA, Cohen SA, Tarvin TL. Rapid analysis of amino acids using pre-
column derivatization. J Chromatogr 1984;336:93–104.
[3] Bradford MM. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding. Anal
Biochem 1976;72:248–54.
[4] Cheung HS, Wang FL, Ondetti MA, Sabo EF, Cushman DW. Binding of peptide
substrates and inhibitors of angiotensin-converting enzyme. Importance of the
COOH-terminal dipeptide sequence. J Biol Chem 1980;255:401–7.
[5] Clare DA, Swaisgood HE. Bioactive milk peptides: a prospectus. J Dairy Sci
2000;83:1187–95.
[6] Coppey LJ, Davidson EP, Rinehart TW, Gellett JS, Oltman CL, Lund DD, et al. ACE
inhibitor or angiotensin II receptor antagonist attenuates diabetic neuropathy
in streptozotocin-induced diabetic rats. Diabetes 2006;55:341–8.
[7] Corradi HR, Chitapi I, Sewell BT, Georgiadis D, Dive V, Sturrock ED, et al.
The structure of testis angiotensin-converting enzyme in complex with the
C domain-specific inhibitor RXPA380. Biochemistry (Mosc) 2007;46:5473–8.
[8] Corradi HR, Schwager SL, Nchinda AT, Sturrock ED, Acharya KR. Crystal struc-
ture of the N domain of human somatic angiotensin I-converting enzyme
provides a structural basis for domain-specific inhibitor design. J Mol Biol
2006;357:964–74.
[9] Dixon M. The graphical determination of Km and Ki. Biochem
J
1972;129:197–202.
[10] Erdmann K, Cheung BWY, Schröder H. The possible roles of food-derived bioac-
tive peptides in reducing the risk of cardiovascular disease. J Nutr Biochem
2008;19:643–54.
[11] FitzGerald RJ, Meisel H. Milk protein-derived peptide inhibitors of angiotensin-
I-converting enzyme. Br J Nutr 2000;84(Suppl. 1):S33–7.
[12] Gobbetti M, Ferranti P, Samacchi E, Goffredi F. Production of angiotensin-
1-converting enzyme inhibitory peptides in fermented milks started by
Lactobacillus delbruekii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cre-
moris FT5. Appl Environ Microbiol 2000;66:3898–904.
[13] Guang C, Phillips RD. Plant food-derived angiotensin I converting enzyme
inhibitory peptides. J Agric Food Chem 2009;57:5113–20.
5. Conclusion
[14] Guyton AC, John EH. Text book of human physiology. W.B. Saunders Co.; 2006.
p. 228.
Inhibiting ACE by food protein derived peptides to treat hyper-
tension appears to be attractive because of the abrogation of
adverse side effects normally accompanying synthetic drugs. A
number of food peptides either derived using enzymes or through
processing are known to reduce blood pressure. The concerted
[15] Hong F, Ming L, Yi S, Zhanxia L, Yongquan W, Chi L. The antihypertensive effect
of peptides: a novel alternative to drugs? Peptides 2008;29:1062–71.
[16] Hyoung Lee D, Ho Kim J, Sik Park J, Jun Choi Y, Soo Lee J. Isolation and character-
ization of a novel angiotensin I-converting enzyme inhibitory peptide derived
from the edible mushroom Tricholoma giganteum. Peptides 2004;25:621–7.

1176
V.K. Jimsheena, L.R. Gowda / Peptides 31 (2010) 1165–1176
[17] Ichimura T, Hu J, Aita DQ, Maruyama S. Angiotensin I-converting enzyme
inhibitory activity and insulin secretion stimulative activity of fermented fish
sauce. J Biosci Bioeng 2003;96:496–9.
[18] Jimsheena VK, Gowda LR. Colorimetric, high-throughput assay for screening
Angiotensin I-converting enzyme inhibitors. Anal Chem 2009;81:9388–94.
[19] Kobayashi Y, Yamauchi T, Katsuda T, Yamaji H, Katoh S. Angiotensin-I convert-
ing enzyme (ACE) inhibitory mechanism of tripeptides containing aromatic
residues. J Biosci Bioeng 2008;106:310–2.
[20] Laemmli UK. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 1970;227:680–5.
[21] Lineweaver H, Burk D. The determination of enzyme dissociation constants. J
Am Chem Soc 1934;56:658–66.
[35] Riordan JF. Angiotensin-I-converting enzyme and its relatives. Genome Biol
2003;4:225.
[36] Rutherfurd-Markwick KJ, Moughan PJ. Bioactive peptides derived from food. J
AOAC Int 2005;88:955–66.
[37] Ryan JW, Ryan US, Schultz DR, Whitaker C, Chung A. Subcellular localiza-
tion of pulmonary antiotensin-converting enzyme (kininase II). Biochem J
1975;146:497–9.
[38] Schechter I, Berger A. On the size of the active site in proteases. I. Papain.
Biochem Biophys Res Commun 1967;27:157–62.
[39] Shetty KJ, Rao MS. Studies on groundnut proteins. III. Physicochemical prop-
erties of arachin prepared by different methods. Anal Biochem 1974;62:
108–20.
[22] Liu Li-na, Zhang Sheng-hua, He Dong-ping. Detection of an angiotensin con-
verting enzyme inhibitory peptide from peanut protein isolate and peanut
polypeptides by Western blot and dot blot hybridization. Eur Food Res Technol
2009;230:89–94.
[23] López-Fandin˜o R, Otte J, van Camp J. Physiological, chemical and techno-
logical aspects of milk-protein-derived peptides with antihypertensive and
ACE-inhibitory activity. Int Dairy J 2006;16:1277–93.
[40] Shetty KJ, Rao MS. Studies on groundnut proteins. V. Physico-chemical prop-
erties of arachin prepared by different methods. Anal Biochem 1976;73:
458–70.
[41] Singh B. PepBuild: a web server for building structure data of peptides/proteins.
Nucleic Acids Res 2004;32:W559–61.
[42] Sturrock ED, Natesh R, van Rooyen JM, Acharya KR. Structure of angiotensin
I-converting enzyme. Cell Mol Life Sci 2004;61:2677–86.
[24] Majumder K, Wu J. Angiotensin I converting enzyme inhibitory peptides from
simulated in vitro gastrointestinal digestion of cooked eggs. J Agric Food Chem
2009;57:471–7.
[25] Mallikarjun Gouda KG, Gowda LR, Rao AG, Prakash V. Angiotensin I-converting
enzyme inhibitory peptide derived from glycinin, the 11S globulin of soybean
(Glycine max). J Agric Food Chem 2006;54:4568–73.
[26] Naqvi N, Liu K, Graham RM, Husain A. Molecular basis of exopeptidase activity
in the C-terminal domain of human angiotensin I-converting enzyme: insights
into the origins of its exopeptidase activity. J Biol Chem 2005;280:6669–75.
[27] Natesh R, Schwager SL, Evans HR, Sturrock ED, Acharya KR. Structural
details on the binding of antihypertensive drugs captopril and enalaprilat
to human testicular angiotensin I-converting enzyme. Biochemistry (Mosc)
2004;43:8718–24.
[28] Natesh R, Schwager SL, Sturrock ED, Acharya KR. Crystal structure of the human
angiotensin-converting enzyme–lisinopril complex. Nature 2003;421:551–4.
[29] Nchinda AT, Chibale K, Redelinghuys P, Sturrock ED. Synthesis and molec-
ular modeling of a lisinopril-tryptophan analogue inhibitor of angiotensin
I-converting enzyme. Bioorg Med Chem Lett 2006;16:4616–9.
[30] Patchett AA, Harris E, Tristram EW, Wyvratt MJ, Wu MT, Taub D, et al. A new
class of angiotensin-converting enzyme inhibitors. Nature 1980;288:280–3.
[31] Pina AS, Roque AC. Studies on the molecular recognition between bioactive
peptides and angiotensin-converting enzyme. J Mol Recognit 2009;22:162–8.
[32] Prakash V, Rao MSN. Physicochemical properties of oilseed proteins. CRC Crit
Rev Biochem 1986;20:265–363.
[33] Pripp AH, Isaksson T, Stepaniak L, Sørhaug T. Quantitative structure–activity
relationship modelling of ACE-inhibitory peptides derived from milk proteins.
Eur Food Res Technol 2004;219:579–83.
[43] Thomsen R, Christensen MH. MolDock: a new technique for high-accuracy
molecular docking. J Med Chem 2006;49:3315–21.
[44] Tombs MP. An electrophoretic investigation of Groundnut proteins: the struc-
ture of arachin A and B. Biochem J 1965;96:119.
[45] Tzakos AG, Gerothanassis IP. Domain-selective ligand-binding modes and
atomic level pharmacophore refinement in angiotensin I converting enzyme
(ACE) inhibitors. Chem BioChem 2005;6:1089–103.
[46] van der Ven C, Gruppen H, de Bont DBA, Voragen AGJ. Optimisation of the
angiotensin converting enzyme inhibition by whey protein hydrolysates using
response surface methodology. Int Dairy J 2002;12:813–20.
[47] Vercruysse L, Van Camp J, Morel N, Rouge P, Herregods G, Smagghe G. Ala-
Val-Phe and Val-Phe: ACE inhibitory peptides derived from insect protein
with antihypertensive activity in spontaneously hypertensive rats. Peptides
2010;31:482–8.
[48] Vermeirssen V, Van Camp J, Verstraete W. Bioavailability of angiotensin I con-
verting enzyme inhibitory peptides. Br J Nutr 2004;92:357–66.
[49] Wu J, Aluko RE, Nakai S. Structural requirements of angiotensin I-converting
enzyme inhibitory peptides: quantitative structure–activity relationship study
of di- and tripeptides. J Agric Food Chem 2006;54:732–8.
[50] Wu J, Ding X. Characterization of inhibition and stability of soy protein-
derived angiotensin 1-converting enzyme inhibitory peptides. Food Res Int
2002;35:367–75.
[51] Wu J, Ding X. Hypotensive and physiological effect of angiotensin convert-
ing enzyme inhibitory peptides derived from soy protein on spontaneously
hypertensive rats. J Agric Food Chem 2001;49:501–6.
[52] Yamamoto N, Takano T. Antihypertensive peptides derived from milk proteins.
Nahrung 1999;43:159–64.
[34] Quist EE, Phillips RD, Saalia FK. Angiotensin converting enzyme inhibitory activ-
ity of proteolytic digests of peanut (Arachis hypogaea L.) flour. LWT – Food Sci
Technol 2009;42:694–9.
[53] Yoshikawa M, Fujita H, Matoba N, Takenaka Y, Yamamoto T, Yamauchi R, et
al. Bioactive peptides derived from food proteins preventing lifestyle-related
diseases. BioFactors 2000;12:143–6.