A variant peptide of buffalo colostrum β-lactoglobulin inhibits angiotensin I-converting enzyme activity

Article abstract of DOI:10.1016/j.ejmech.2012.03.057

β-lactoglobulin is a rich source of bioactive peptides. The LC-MS separated tryptic peptides of buffalo colostrum β-lactoglobulin (BLG-col) were computed based on MS-MS fragmentation for de novo sequencing. Among the selected peptides (P1-P8), a variant was detected with methionine at position 74 instead of glutamate. The sequences of two peptides were identical to hypocholesterolemic peptides whereas the remaining peptides were in accordance with buffalo milk β-lactoglobulin. Comparative sequence analysis of BLG-col to milk β-lactoglobulin was carried out using CLUSTALW2 and a molecular model for BLG-col was constructed (PMDB ID-PM0076812). The synthesized variant pentapeptide (IIAMK, m/z-576 Da) was found to inhibit angiotensin I-converting enzyme (ACE) with an IC₅₀ of 498 ± 2 μM, which was rationalized through docking simulations using Molgrow virtual docker.

Full text of DOI:10.1016/j.ejmech.2012.03.057

European Journal of Medicinal Chemistry 53 (2012) 211e219
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A variant peptide of buffalo colostrum b-lactoglobulin inhibits angiotensin
I-converting enzyme activity
,
A.C. Rohit a, K. Sathisha b, H.S. Aparna a
*
a Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India
b Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
b
-lactoglobulin is a rich source of bioactive peptides. The LC-MS separated tryptic peptides of buffalo
Received 19 September 2011
Received in revised form
29 March 2012
Accepted 30 March 2012
Available online 6 April 2012
colostrum -lactoglobulin (BLG-col) were computed based on MSeMS fragmentation for de novo
sequencing. Among the selected peptides (P1eP8), a variant was detected with methionine at position 74
instead of glutamate. The sequences of two peptides were identical to hypocholesterolemic peptides
b
whereas the remaining peptides were in accordance with buffalo milk
b-lactoglobulin. Comparative
sequence analysis of BLG-col to milk -lactoglobulin was carried out using CLUSTALW2 and a molecular
b
model for BLG-col was constructed (PMDB ID-PM0076812). The synthesized variant pentapeptide
(IIAMK, m/z-576 Da) was found to inhibit angiotensin I-converting enzyme (ACE) with an IC50 of
Keywords:
Bubalus bubalis
Colostrum
498 ꢀ 2
m
M, which was rationalized through docking simulations using Molgrow virtual docker.
Ó 2012 Elsevier Masson SAS. All rights reserved.
b
-Lactoglobulin
De novo sequencing
Angiotensin I-converting enzyme
1. Introduction
lactation mainly in ruminants and other mammalian species except
primates [8]. The functional advantage of
cated in the transport of vitamin D [9] and upon enzymatic diges-
tion, the peptides of -lactoglobulin are known to play pivotal role
in human health and modulate several regulatory processes [10].
The peptides effectively exhibit antihypertensive [11], antimicrobial
[12], antioxidant [13,14] and also immunostimulatory effects [15].
b-lactoglobulin is impli-
Milk, a mammalian specific biological fluid, gains considerable
attention as its proteins and peptides exert a diverse range of
nutritional and functional activities. Colostrum, the early lactation,
has a nutritional profile and immunological composition that
substantially differs from that of mature milk. It possesses major
b
milk proteins like
b-lactoglobulin,
a
-lactalbumin, glycomacropep-
The peptides of b-lactoglobulin can suppress the cholesterol
tides, albumin proteose peptone along with immunoglobulins [1]
and array of growth factors [2e4]. Recent studies suggest that
colostral fractions, or individual peptides present in colostrum are
useful for the treatment of a wide variety of gastrointestinal disor-
ders including inflammatory bowel disease, nonsteroidal anti-
inflammatory drug (NSAID) induced gut injury and chemotherapy-
induced mucoscitis [5]. To date, several bioactive peptides have
been identified in protein hydrolysates and fermented foods of milk
but not of colostrum [6,7]. The multifunctional property associated
with milk bioactive peptides includes antihypertensive, antioxidant,
hypocholesterolaemic, opioid, antimicrobial, immunomodulatory
and cytomodulatory activities [4].
absorption as evidenced in Caco-2 cells, a model system used to
study lipid metabolism. Furthermore, hypocholesterolemic activity
exhibited by a tetrapeptide was even greater than the medicine
sitosterol as studied in rats [16]. The high nutritional and functional
value of -lactoglobulin has therefore made it a preferred food
b-
b
ingredient in formulation of modern food supplement, BioZate [4].
Angiotensin I-converting enzyme (ACE) has a critical role in
cardiovascular function by cleaving the C-terminal His-Leu dipep-
tide from angiotensin I to produce a vasoconstrictor angiotensin II.
In the global scenario, inhibition of ACE is considered as one of the
potential tasks in treating high blood pressure, heart failure, dia-
betic nephropathy, and type 2 diabetes mellitus. Consequently,
synthetic ACE inhibitors such as lisinopril, captopril, enalapril and
alecepril are effectively used in the treatment of hypertension
despite their side effects. Hence, bioactive antihypertensive
peptides of food origin are increasingly gaining importance rather
than synthetic drugs in hypertension therapy. Although casein
derived peptides from milk have been reported to have ACE
Of the different proteins present in milk,
b-lactoglobulin is
a major soluble globular protein containing 162 amino acids
secreted in the mammary gland during late pregnancy and
* Corresponding author. Tel.: þ91 821 2419883; fax: þ91 821 2419363.
E-mail address: hsa.uom@gmail.com (H.S. Aparna).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.057

212
A.C. Rohit et al. / European Journal of Medicinal Chemistry 53 (2012) 211e219
inhibitory activity [17], the peptides from whey proteins are less
studied and characterized except for -lactoglobulin. Many
peptides of -lactoglobulin derived after treatment with trypsin,
chymotrypsin, pepsin, thermolysin and proteinase K have been
identified to possess moderate to high ACE inhibitory activity [13].
(P4), m/z-818.62þ (P5), m/z-851.52þ (P6) and m/z-772.23þ (P7).
Except for P1 (Fig. 2, Table 1), the masses of all other peptides match
the expected masses of BLG-col tryptic peptides. Hence, molecular
ion m/z-5761þ along with other peptides was analyzed to deter-
mine the sequence information. This suggests the presence of iso-
b
b
Realizing the biomedical importance of
b-lactoglobulin, we have
forms of b-lactoglobulin with possible sequence variations; as such
purified -lactoglobulin (BLG-col) from buffalo (Bubalus bubalis)
b
information is not available either from buffalo milk or colostrum.
colostrum for the first time [18]. Buffalo, a major milking ruminant
in the Asian countries, predominant in India contributes to 53% of
2.1.1. De novo sequencing of BLG-col peptides
the total bubaline population of the world.
b
-lactoglobulin, a major
The protonated molecular ion (m/z-576.5) detected after 8.3 min
by LC-MS showed variation to the PMF of BLG-col [18] and tryptic
whey protein has been extensively studied from bovine milk but
there is limited information from buffalo milk and no reports from
buffalo colostrum. In the present study, we have employed LC-MS/
MS with Collision Induced Dissociation (CID) and Electron Transfer
Dissociation (ETD) based fragmentation on a 3D radio frequency
(rf) ion trap [19e21] to unravel the sequence similarity and/or
peptides of bovine milk
b-lactoglobulin. As shown in Fig. 2, the
molecular ion of m/z-573.3 in bovine milk
b
-lactoglobulin was
detected as 576.5 in BLG-col and hence, MS/MS analysis was carried
out for this ion to unravel the sequence information. The CID based
MS/MS fragmentation revealed almost complete b- and y-ion series
corresponding to m/z-576.5 (M þ Hþ) with “AMK/Q” motif (b2eb5)
and “AI/L” motif (y2ey4) and the resultant sequence was I/LAMK/Q
(Mr calc. ¼ 575.35, Fig. 3A). Submission of .mgf file to Mascot MS/
variation of BLG-col to buffalo and bovine milk
b-lactoglobulin.
Further, in vitro and in silico analysis validated the ACE inhibitory
activity of a variant peptide detected in BLG-col for its possible use
as a potent drug candidate in hypertension therapy.
MS ion search database did not show any match to b-lactoglobulin
sequence although it has close match to hypocholesterolemic
peptide “IIAEK” from bovine milk -lactoglobulin. This was further
2. Results and discussion
b
supported by the identification of b4 and y2 ions with 429.4 and
278.2 for “IIAMK” instead of 427.25 and 276.15 for “IIAEK” respec-
tively (Supplementary file: Table 1). Thus, we could identify single
amino acid variation in P1 with methionine at position 74 instead of
glutamate between the CID derived sequence data of the present
The “early” milk, which is known as ‘colostrum’ produced by
mammals during early lactation, is exceptionally complex in its
composition. The health promoting properties of the most abun-
dant bovine milk
whereas no such detailed information is available from colos-
trum. Hence, we have purified -lactoglobulin (BLG-col) to homo-
geneity from buffalo colostrum [18] and used it in the present study
to delineate sequence similarity and/or variation between
b-lactoglobulin has been well documented [13]
study and the available sequence for bovine and buffalo milk
b-
b
lactoglobulin in the database. Although bovine -lactoglobulin is
b
known to express in multiple forms with one or two amino acid
variations [23], none of the isoforms have been identified in buffalo
b
-
lactoglobulin derived from colostrum and milk employing tandem
mass spectrometry and to determine its functional property.
b
-lactoglobulin. But, Vohra et al. [22] have reported the presence of
genetic variants of -lactoglobulin gene and studied its association
b
with milk composition traits in riverine buffalo. The single amino
acid variation identified in the present study could be a genetic
variant. Incidentally, the sequence tag refers to a potent candidate
molecule that decreases the absorption of cholesterol in compar-
ison to well-known drugs as evidenced by both in vivo and in vitro
studies [16]. Hence, biological implication of the deduced sequence
offers further investigation.
2.1. Chemistry
The pure protein was subjected to in-solution trypsin digestion
and the peptide mass fingerprint (PMF) of MS analysis revealed
a match of 67% to b-lactoglobulin of buffalo and bovine milk [18].
The LC-ESI-MS/MS analysis was performed with CID and ETD
capabilities to derive the sequence information of the tryptic
peptides of BLG-col. The separation of peptides by LC, facilitated
detection of 4, 2 and 1, single, double and triple charged ion pairs
respectively (P1eP7, Fig. 1, Table 1) in analogy to the PMF of the
BLG-col [18]. Accordingly, the identified protonated molecular ions
were m/z-5761þ (P1), m/z-6741þ (P2), m/z-9161þ (P3), m/z-9331þ
The CID based MS/MS analysis of other single charged ions
(P2eP4: Fig. 3BeD; Supplementary files: Tables 2e4) did not reveal
any variations and were in accordance with the
buffalo milk [24] and cDNA sequence [25]. Further, the sequence
deduced for P4 represented N-terminus of -lactoglobulin and
precisely matched to the N-terminal sequence data and PMF [18]
b-lactoglobulin of
b
Fig. 1. LC-MS analysis of tryptic peptides derived from BLG-col. Insert letters P1eP7 denotes single [M þ H]þ-576, [M þ H]þ-673, [M þ H]þ-916, [M þ H]þ-933, double [M þ H]þ-818
(m/z-1636), [M þ H]þ-851 (m/z-1702) and triple [M þ H]þ-772 (m/z-2313) charged ions.

A.C. Rohit et al. / European Journal of Medicinal Chemistry 53 (2012) 211e219
213
Table 1
2.1.2. Multiple sequence alignment and protein modeling of BLG-col
MHþ Average mass of tryptic peptides derived from the BLG-col after trypsin
digestion by LC-CID-MS/MS analysis.
The sequences (P1eP8) thus obtained for BLG-col following de
novo sequencing strategy was analyzed employing the multiple
sequence alignment program CLUSTAL-W2 against buffalo and
Peptides
(position)
Retention
Precursor ions
Mr
Mr
time (min)
m/z (Da)
(expect)
(observed)
bovine milk b-lactoglobulin. Upon sequence comparison, variations
P1 (71e75)
P2 (9e14)
P3 (84e91)
P4 (1e8)
P5 (125e138)
P6 (149e162)
P7 (41e60)
8.3
8.2
7.7
7.6
7.3
7.5
7.2
576.5þ
674.5þ
916.5þ
933.6þ
1636.7
1702.5
2313.6
573.3þ
674.5þ
916.5þ
933.6þ
1636.7
1702.5
2313.6
576.5þ
674.5þ
916.5þ
933.6þ
818.62þ
851.52þ
772.23þ
at position 74 (M/E) of BLG-col to both buffalo and bovine milk
b
-
-
lactoglobulin and N-(I/L) and C-(V/I) terminus of bovine milk
b
lactoglobulin were discrete (Fig. 6). Eventually, the molecular
model for BLG-col was constructed using bovine milk -lacto-
b
globulin sequence as template and uploaded to PMDB with ID-
PM0076812 (http://mi.caspur.it/PMDB/user/search.php).
2.1.3. Characterization of synthetic peptide
while, sequences of P2 and P3 matched to 9e14 and 84e91
sequence tags of bovine and buffalo milk -lactoglobulin [24,25].
The variant peptide (IIAMK) detected in the BLG-col was
synthesized by solid phase peptide synthesis using wang resin
(Fmoc chemistry) and purified by RP-HPLC. The sequence of the
synthetic peptide was validated by LC-MS/MS, which showed
complete match to the variant peptide deduced in the present
study by de novo sequencing (Supplementary file: Fig. 1).
b
The double (m/z-818.6 and 851.5) and triple charged (m/z-772.2)
ions were also analyzed by MS/MS analysis in CID and ETD capa-
bilities. Integration of CID generated product ion spectrum with ETD
fragmentation (Fig. 4AeC; Supplementary files: Tables 5e7) showed
complete sequence homology to 125e138 (P5), 149e162 (P6) and
41e60 (P7) amino acid sequence of buffalo milk
the sequence of P6 that matches the C-terminal sequence of buffalo
milk -lactoglobulin, differs from bovine -lactoglobulin by having
b-lactoglobulin. But
2.2. Biological studies
b
b
valine residue instead of isoleucine [24e26].
2.2.1. ACE inhibitory activity
We have analyzed yet another peptide that corresponds to the
experimental mass of 2708.404 Da following MALDI-LIFT MS/MS
technology, which was not recognized by the LC-MS/MS. The
manual sequencing of the peptide using b- and y-ion series yielded
“VAGTWYSL/IAMAASDI/LSL/IL/IDAQ/KSAPL/IR” as shown in Fig. 5
(Supplementary file: Table 8). The Mascot MS/MS ion search
against NCBI database confirmed its identity with the sequence tag
15e40 (P8) of buffalo milk b-lactoglobulin and deposited in NCBI
database under accession no. PSE130 (http://www.ncbi.nlm.nih.
gov/peptidome).
Fig. 7 explicit the action of the synthetic variant peptide on ACE
in a dose dependent manner with IC50 of 498 ꢀ 2
was comparatively higher than the ACE inhibitory peptides repor-
ted for the tryptic peptides of -lactoglobulin except for YLLF and
mM. The IC50 value
b
ALPMHIR sequences [27]. Cheung et al. [28] suggested that the
peptides with branched chain amino acids at amino and Pro at
carboxy-terminus are the most favored for ACE inhibitory activity.
In addition, presence of Lys or Arg as C e terminal residue would
also enhance the efficiency of ACE inhibition [13]. Since an under-
standing of the pentapeptide inhibitory binding mode to ACE has
Fig. 2. LC-MS spectra of [M þ H]þ-573.3 and 576.5 of milk
b
-lactoglobulin and BLG-col. [M þ H]þ-576.5 of BLG-col was analyzed by CID.

214
A.C. Rohit et al. / European Journal of Medicinal Chemistry 53 (2012) 211e219
Fig. 3. LC-CID MS/MS spectra of P1eP4. Matched b- and y-ions for [M þ H]þ-576 (A), 674 (B), 916 (C) and 933 (D). Inset: Sequence information based on fragmentation pattern.
many potential applications, we studied interaction of the variant
peptide with human tACE catalytic site and compared with the
known drug, lisinopril through molecular docking.
while, the peptide IIAMK formed hydrogen bonds with all the
active site residues except for Lys-511 and Glu-162 of S2 and S10
respectively (Fig. 8). Subsequently, the effectiveness of pentapep-
tide in the Molegro docking programme was evaluated by docking
the lisinopril and the resultant geometry was compared with the
corresponding crystal structure of tACE-lisinopril complex (PDB:
1O86). The best returned pose in presence of Zn (II) with the
docking score of 151.51 for the peptide further confirmed the
involvement of stabilizing forces like hydrogen bond, electrostatic
and hydrophobic interactions in the enzymeepeptide interaction
(Fig. 8). These results clearly show the interaction of the peptide
with the active site residues of ACE as validated by docking
calculations. Furthermore, our results also indicate that the
binding mode may be different for the previously reported
potential ACE inhibitory peptides of milk proteins and casein
[17,30].
2.2.2. Molecular docking
The variant peptide (IIAMK, m/z-576), with hydrophobic amino
acids Ile-Ile-Ala as N-terminal sequence and a positively charged
Lys residue at the C-terminal position, was used to examine its
effect on ACE activity by docking analysis and further substantiate
the results obtained by in vitro ACE inhibition assay. The main
interacting residues at the ACE active site are divided into three
pockets as described by Pina and Roque [29] and accordingly, Ala-
354, Glu-384 and Tyr-523 correspond to S1 pocket, while S2
pocket includes Gln-281, His-353, Lys-511, His-513 and Tyr-520
residues and S1’ contains Glu-162 residue. As shown in Table 2,
ACE inhibitor lisinopril interacted with all the active site residues

A.C. Rohit et al. / European Journal of Medicinal Chemistry 53 (2012) 211e219
215
Fig. 4. LC-CID/ETD-MS/MS spectra of P5eP7. Matched b-/y-, c-/z-ions and peptide sequence for [Mþ2H]2þ -818(A), [Mþ2H]2þ -851(B) and [Mþ3H]3þ -772(C).
3. Conclusion
promising natural food additive in designer foods to overcome the
side effects of synthetic drugs.
The template based de novo sequence analysis of BLG-col was
successfully demonstrated using tandem mass spectrometry and
a variant peptide discovered for the first time with methionine at
position 74 instead of glutamate. The ACE inhibitory activity of the
synthetic peptide along with docking simulations envisages the
functional capability of variant peptide of BLG-col. The branched
chain amino acids at amino and Pro at carboxy-terminus in
peptides favor ACE inhibitory activity [28], and Lys or Arg as C
e terminal residue enhance the efficiency of ACE inhibition [13].
Thus we suppose, a variant pentapeptide of BLG-col having Ile at N-
and Lys as C e terminus residues would be an attractive and
4. Experimental
4.1. Isolation and purification of BLG-col
The whey proteins were isolated from buffalo colostrum as
described earlier [31]. The purification of BLG-col was achieved by
ammonium sulfate precipitation followed by gel permeation
chromatography. The homogeneity and molecular weight was
determined using SDS-PAGE and MALDI-TOF [18].

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A.C. Rohit et al. / European Journal of Medicinal Chemistry 53 (2012) 211e219
Fig. 5. MALDI-TOF MS/MS spectra of P8. Matched b- and y-ions and peptide sequence for 2707.4 Da.
4.2. Protein digestion
C-18 column (4.6 ꢂ 150 mm; ZORBAX RX-C18, 5
mm particle size,
Agilent) using a linear gradient of acetonitrile containing 0.1% for-
Enzyme digestion of BLG-col was performed using trypsin
(Procine trypsin, Promega, USA) in 50 mM NH4HCO3 buffer pH 8.0.
Enzyme to protein ratio was maintained at 1:50 and the reaction
mixture was incubated at 37 ꢁC for 12 h. The tryptic peptides were
extracted using acetonitrile (50%) containing 1% TFA (ꢂ3) and the
extracted peptides were pooled and volume was reduced with
vacuum centrifuge. The isolated tryptic peptides were dissolved in
0.1% TFA (v/v) and an aliquot was desalted using reverse-phase C18
Zip-Tip (Millipore Billerica, MA, USA). The C18 Zip-Tip was
sequentially equilibrated with methanol, TFA (0.1%) before loading
tryptic peptides onto it. After washing with 0.1% TFA, the elution
was carried out in 0.5 ml 80% acetonitrile containing 0.1% TFA [32].
mic acid (5e95%) in 55 min at a flow rate of 200 ml/min. LC was
interfaced directly with an ion trap (HCT ULTRA ETD II, Bruker
Daltonics, Germany) mass spectrometer equipped with two Octa-
pole followed by an ion trap and a separate hexapole next to
negative chemical ionization chamber (nCI). The electrospray
ionization (ESI) MS/MS data of the tryptic peptides were obtained
using CID and ETD. Helium gas was used as the collision gas for
collision induced dissociation (CID) experiments. Fluoranthene was
used as the electron transfer reagent and methane was used for the
chemical ionization. The reaction time of electron transfer was
typically set at 100 ms with the following parameters: compound
stability 100%, with maximum output of ETD reagent ion 202 m/z,
scan range 100e2800 m/z, target mass of 900 m/z and reagent ion
ICC was set to 200,000. The CID-MS/MS data were processed using
Esquire data analysis software, version 4.0 and .mgf (Mascot
Generic Format) file extracted from data analysis software was
analyzed by Mascot MS/MS ion search database.
4.3. LC-CID ETD-MS/MS analyses
HPLC workflow was performed on an Agilent (HP1100 Series)
with detection at 226 nm. The tryptic peptides were separated on
Fig. 6. Multiple sequence alignment of BLG-col sequence derived using LC-CID/ETD-MS/MS and MALDI-MS/MS. BLG_COL refers to buffalo colostrum
LACT_BUB denotes buffalo milk -lactoglobulin sequence and LACT_BOS denotes bovine milk -lactoglobulin sequence. P1eP8 denotes different peptides and their respective
sequences. Red stars indicate amino acid variation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
b-lactoglobulin sequence;
b
b

A.C. Rohit et al. / European Journal of Medicinal Chemistry 53 (2012) 211e219
217
spectral data were processed by Bruker Daltonics Data analysis
(4.0 Version) software [33].
4.5. Multiple sequence alignment
The BLG-col sequence derived from de novo analysis was
analyzed using ClustalW2 against buffalo and bovine milk
b-
lactoglobulin sequences acquired from UniProt database (http://
www.uniprot.org/uniprot/?query¼betalactoglobulin&sort¼score).
4.6. Homology based protein modeling
The homology based protein modeling was carried out using
SWISS-MODEL and SWISS-Pdb Viewer 4.0 (Deep Viewer) software.
SWISS-MODEL was gained access via a web interface at http://
swissmodel.expasy.org or directly as a link from ExPASy server.
The program Deep Viewer 4.0 (SWISS-pdb Viewer) was down-
loaded at http://www.expasy.org/spdbv/. The amino acid sequence
data of BLG-col was entered into the SWISS-MODEL to build
template based protein model. The complete PDB project file was
downloaded and submitted to SWISS-Pdb Viewer for evaluation of
sequence diversity [34].
Fig. 7. The semi logarithmic plot to determine IC50 of variant peptide. X-axis showing
log of variant peptide ( M) and Y-axis showing the % inhibition.
m
4.4. MALDI-TOF MS/MS analysis
4.7. Solid phase peptide synthesis
Tryptic peptides of BLG-col were analyzed by a MALDI-TOF/
TOF tandem mass spectrometer in positive ion reflector mode
using Ultra flex TOF/TOF (Bruker Daltonics, Bremen, Germany)
equipped with nitrogen laser (337 nm). Equal volume of enzyme
digested samples and saturated matrix solution (2,5-
dihydroxybenzoic acid in 50% acetonitrile containing 0.1% TFA)
were mixed and spotted on a MALDI plate and the spectra were
recorded in the linear mode using Bruker Daltonics FLEX control
software and the most intense peptides (up to m/z-1000) were
further accelerated in to the LIFT cell for MS/MS analysis and the
Using standard Fmoc chemistry, acid form of the pentapeptide
was assembled on an automatic peptide synthesizer (Model CS 136,
CS Bio Co. San Carlos, CA, USA). Fmoc-L-Lys-2-chlorotrityl resin was
preloaded with the peptide containing carboxy terminal Lys. The
sequence of pentapeptide was verified by amino terminal
sequencing and amino acid analysis. Semi-preparative HPLC
equipped with
a LC8A pump on a C-18 Shimpak column
(25 cm ꢂ 21.2 mm) 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
Table 2
Docking analysis for ligand lisinopril and pentapeptide using an automated docking software Molgro Virtual Docker 2008, version 3.2.1. Zinc’s active site was shown in the red.
Sl no
Amino acid
residue
Crystal structure with ligand (LPR)
Docking with ligand (LPR)
Docking with PEPTIDE
Atom in
ligand
Atom in
receptor
Distance
(in Å)
Atom in
ligand
Atom in
receptor
Distance
(in Å)
Atom in
ligand
Atom in
receptor
Distance
(in Å)
1
2
3
4
5
6
7
8
Asn-66
Asn-70
e
e
HG21
HG22
ND2
HD22
2.55
4.87
e
e
e
Glu-162
Gln-281
His-353*
Ala-354
Ser-355
Ala-356
Trp-357
Asp-377
Val-380
His-383
Glu-384
His-387
Glu-411
ASP-415
Phe-457
Lys-511
Phe-512
His-513
Val-518
Tyr-520
Arg-522
Tyr-523
Tyr-527
N3
O4
O1
N1
C17
N
OE2
NE2
NE2
O
CB
O2
3.45
3.22
2.76
2.92
3.98
4.95
e
N8
O5
O1
O
C25
N
OE2
NE2
NE2
N6
CB
O2
2.42
2.44
3.17
2.67
3.93
4.46
e
HE2
O
N
H
O
HE22
NE2
O
N
N
3.72
2.53
3.66
4.32
2.82
2.98
e
9
H1
CG
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
N3
C12
O2
O2
O2
O3
OD1
CG1
NE2
OE2
NE2
OE1
4.23
3.99
3.54
2.70
3.33
3.14
e
N8
C18
O2
O2
O2
O3
OD1
CG1
NE2
OE1
NE2
OE1
4.78
4.42
4.16
2.71
3.98
3.80
e
HB2
NE2
N
O
O
CG1
HA
4.65
2.80
2.96
3.25
2.84
4.89
3.77
e
OE2
NE2
OE1
OD1
CZ
HD2
NZ
C6
O4
C17
O1
C21
O4
CZ
NZ
CE2
NE2
CG2
OH
3.72
2.93
3.73
3.11
3.77
2.56
e
C14
O5
C25
O1
C29
O5
CZ
NZ
CZ
NE2
CG2
OH
3.91
2.98
3.36
2.74
4.31
3.38
e
O
CD2
NE2
CG1
OH
NH1
OH
4.77
2.74
4.60
4.45
4.28
2.56
3.74
OH
SD
H92
CB
OH
NZ
O3
C7
OH
CE1
2.77
4.69
O1
H
OH
CE1
3.01
4.19
CE1

218
A.C. Rohit et al. / European Journal of Medicinal Chemistry 53 (2012) 211e219
Fig. 8. Details of the interaction between ACE active site and the inhibitory peptide (Yellow) and lisinopril (Red). The best ranked docking poses are shown in (A) Protein structure of
ACE (PDB ID: 1O86) with binding site of the enzyme was computed within a spacing of x: 37.26, y: 35.35 and z: 46.21 Å with bound peptide and lisinopril (B) Hydrogen bond
interaction of peptide and lisinopril with ACE active site amino acid located surrounding at distance below 4 Å (C) Electrostatic interaction of peptide and lisinopril with ACE active
site amino acids (D) Hydrophobic interaction of peptide and lisinopril with ACE active site amino acids. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
a SPD-M10AVP photodiode array detector at 230 nm. Purity of
peptide was >99% as determined by RP-HPLC.
which represents the human tACE bound to lisinopril at 2 Å reso-
lution [36]. The structure of the peptide was constructed using
automated PepBuild web server [37]. The possibility of binding,
precise location of binding site and mode of binding of the ligand
was carried out using an automated docking software Molgro
Virtual Docker 2008, version 3.2.1 [38]. Possible binding confor-
mations and orientations were analyzed by clustering methods
embedded in MolDock.
All docking studies were carried out using the crystal structure of
tACE complexed with the inhibitor lisinopril (PDB: 1O86) as the
template. All water molecules and inhibitor lisinopril were excluded
whereas zinc and chloride atoms were retained in the active site,
fixed to their crystal position, through out the docking process. The
binding site of the enzyme 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 grid resolution of 0.3 Å. A value of population
size and maximum interactions 100 and 10,000 respectively were
used for each run and 5 best poses were retained for ligand. Lisi-
nopril was docked into the template structure as a positive control
and the root-mean-square deviation (RMSD) was obtained.
4.8. Biology
4.8.1. In vitro ACE inhibition assay
ACE activity was assayed by monitoring the release of Hippuric
acid (HA) from the substrate Hippuryl-Histidyl-Leucine (HHL) as
described by Jimsheena et al. [35]. The assay mixture contained
0.125 ml of 50 mM sodium borate buffer pH 8.2 containing 0.3 M
NaCl, 0.05 ml of 5 mM HHL and 25 ml of ACE enzyme prepared from
Rat kidney. The reaction mixture was incubated at 37 ꢁC for 30 min
and stopped by adding 0.2 ml 1 M HCl followed by 0.4 ml of pyri-
dine and 0.2 ml of Benzene sulfonyl chloride (BSC). The color
developed was measured at 410 nm in a UVeVIS spectrophotom-
eter (Shimadzu). One unit of ACE activity was defined as the
amount of enzyme, which released 1 m
mole of HA per min at 37 ꢁC.
ACE pre incubated with different concentrations of the peptide
fractions and the IC50 was determined by plotting log peptide
concentration on X-axis and % inhibition on Y-axis. The percentage
inhibition curves were plotted using minimum of five determina-
tions and IC50 values computed from the semi logarithmic plots.
Acknowledgments
4.8.2. Docking analysis
The model for ACE used in this study was derived from the co-
ordinates of the structure labeled 1O86 in the protein data bank,
We thank University Grant Commission, New Delhi, India for the
grant (F31-294/2005-06) to undertake this investigation and Rohit
A. Chougule thank Indian Council of Medical Research, New Delhi

A.C. Rohit et al. / European Journal of Medicinal Chemistry 53 (2012) 211e219
219
[18] R.A. Chougule, H.S. Aparna, Characterization of
b
-lactoglobulin from buffalo
for grant of Senior Research Fellowship (45/12/2009-Biochemistry/
BMS).
(Bubalus bubalis) colostrum and its possible interaction with erythrocyte
lipocalins-interacting membrane receptor, J. Biochem. 150 (2011) 279e288.
[19] L.M. Mikesh, B. Ueberheide, A. Chi, J.J. Coon, J.E. Syka, J. Shabanowitz,
D.F. Hunt, The utility of ETD mass spectrometry in proteomic analysis, Bio-
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Peptide and protein sequence analysis by electron transfer dissociation mass
spectrometry, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 9528e9533.
[21] N.D. Udeshi, P.D. Compton, J. Shabanowitz, D.F. Hunt, K.L. Rose, Methods for
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