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One Peptide Derived from Hen Ovotransferrin as Pro-drug
to Inhibit Angiotensin Converting Enzyme
NAI-YUAN LEE1, JUEI-TANG CHENG2*, TOSHIKI ENOMOTO3 AND YOSHIHISA NAKANO4
1. Livestock Research Institute, Tainan County 712, Taiwan, R.O.C.
2. Department Pharmacology, College of Medicine, National Cheng Kung University, Tainan City 701, Taiwan, R.O.C.
3. Department of Food Science, Ishikawa Prefectual University, Nonoichi, Ishikawa 921-8836, Japan
4. Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
(Received: September 7, 2005; Accepted: November 22, 2005)
ABSTRACT
Angiotensin Ι-converting enzyme (ACE) inhibitory peptide was derived from hen ovotransferrin and identified as Lys-Val-Arg-
Glu-Gly-Thr-Thr-Tyr. It produced a concentration-dependent inhibition of ACE activity in vitro with an IC50 value of 102.8 μM.
After hydrolysis by ACE, the product (Lys-Val-Arg-Glu-Gly-Thr) has an IC50 value of 9.1 μM that was about 11-fold lower of the
parent peptide. Thus, Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr can be considered as a pro-drug. Moreover, Lys-Val-Arg-Glu-Gly-Thr-
Thr-Tyr and Lys-Val-Arg-Glu-Gly-Thr were intravenously administered into spontaneously hypertensive rats (SHR) to monitor the
time-course change of systolic blood pressure. We found that Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr and its hydrolyzed product produced
the maximal reduction of systolic blood pressure at 40 min and 20 min after injection, respectively. This 20 min delay might be
considered as the time required for conversion of prodrug into Lys-Val-Arg-Glu-Gly-Thr, the true ACE inhibitor. In conclusion, the
obtained results suggest that Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr works as the pro-drug of Lys-Val-Arg-Glu-Gly-Thr to inhibit ACE
activity in vivo.
Key words: angiotensin-converting enzyme (ACE) inhibitor, antihypertensive peptide, hen ovotransferrin
* Author for correspondence. Tel: +886-6-2372706;
Fax: + 886-6-2386548; E-mail: jtcheng@mail.ncku.edu.tw
藥物食品分析 第十四卷 第一期
INTRODUCTION
The renin-angiotensin system is well known to
regulate blood pressure in the circulatory system. Actually,
angiotensin I-converting enzyme (ACE) (dipeptidyl
carboxypeptidase, EC 3.4.15.1) plays an important role
in this renin-angiotensin system. Captopril and enalapril
are known as antihypertensive drugs by retarding the
catalytic action of ACE. Thus, ACE inhibitors exhibit
antihypertensive activity in spontaneously hypertensive
rats (SHR) and hypertensive patients(1). Recently, many
ACE inhibitory peptides were derived from food such as
egg yolks(2), sh muscle(3-5), wakame(6), sour milk(7) and
dry bonito(8).
Ovotransferrin, an iron-binding glycoprotein
belonging to the transferrin family protein, was found to
contain 13% of the total protein in egg white. Lys-Val-
Arg-Glu-Gl y- Thr- Thr- Tyr iso la ted from chymotryp ti c
hydrolysates of ovotransferrin has ACE inhibitory
activity. Also, it can lower blood pressure in SHR after
an intravenous injection. In the present study, using the
preincubation method(3), Lys-Val-Arg-Glu-Gly-Thr-Thr-
Tyr was investigated and characterized as a true ACE
inhibitor or a pro-drug peptide.
MATERIALS AND METHODS
I. Chemicals
Peptide for assay of the ACE inhibitory activity was
ordered to prepare using Fmoc amino acid active derivatives
from Sigma-Genosys (TX, USA). ACE (EC 3.4.15.1)
and hippuryl-histidyl-leucine were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). Other reagents used
were of analytical grade.
II. Determination of the Stability of Peptide for ACE
Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr (0.02 mM) was
incubated with 28 mU of ACE at 37°C for 3 hr, and the
reaction was stopped by boiling for 10 min, as described
previously(8). In order to check the stability of the tested
inhibitory peptide for ACE, the reaction was immediately
applied to a RP-HPLC system, using a LiChroCART
C18 column (4 mm I.D. × 250 mm, a product of Merck,
Frankfurter City, Germany) and a mixture of solvent
A (0.1% trifluoroacetic acid in water) and B (0.1%
trifluoroacetic acid in acetonitrile). A linear gradient of A
and B (0 to 67% B) was applied to the column for 18 min at
a ow rate of 1 mL/min and the absorbance of the eluate at
214 nm was monitored.
31
藥物食品分析 第十四卷 第一期
Journal of Food and Drug Analysis, Vol. 14, No. 1, 2006, Pages 31-35
Journal of Food and Drug Analysis, Vol. 14, No. 1, 2006
32
III. ACE Inhibitory Activity between Digests with ACE and
Puried Peptide
IC50 values (concentration of testing agent required to
inhibit 50% of the ACE activity) of Lys-Val-Arg-Glu-Gly-
Thr-Thr-Tyr before and after incubation with ACE were
compared. Moreover, fractions due to the digests with ACE
applying RP-HPLC system were collected and the sequence
of peptides was determined using an Applied Biosystems
gas-phase sequencer 492 Protein Sequencer (Applied
Biosystems Inc., Foster City, CA, USA). The acetonitrile in
fraction was removed using a centrifugal evaporator and the
sample was dissolved in water and neutralized by adding an
alkaline solution for the assay of ACE inhibitory activity.
The sample was assayed in vitro for the ability to
inhibit ACE activity according to the previous method(9). In
brief, 100 μL of 4.7 mM hippuryl-L-histidyl-L-leucine/300
mM NaCl/400 mM phosphate buffer solution (pH 8.5)
was added using 50 μL of testing sample or vehicle used
to dissolve the testing sample. Then, 100 μL (2.5 mU) of
ACE/distilled water was mixed with the above substrate
solution to initiate the reaction that was carried out by
incubation in a water bath at 37 ± 1°C under shaking for 60
min. Finally, 1.5 mL of 0.3 M sodium hydroxide was added
to terminate the reaction. The formed histidyl-leucine was
then labeled by 100 μL of 2% phthaldialdehyde/methanol
at room temperature for 10 min and the reaction was
terminated by 200 μL of 3 M HCl. The formed uorescence
compound was diluted using distilled water to 250 times
and the fluorescence intensity was then estimated by a
spectrofluorometer (EX340, EM455; Hitachi, F-3000).
Substrate with distilled water only was used as the blank,
while the mixture without testing sample but the same
volume of distilled water was treated as control. The
inhibitory ratio (%) of ACE was calculated as (C - A)/(C -
B) × 100%, where A is absorbance of the testing sample,
B is absorbance of the blank and C is absorbance of the
control. Sample was tested at ve concentrations to obtain
the standard curve for the determination of the IC50 value.
Sample was tested in triplicate.
IV. Determination of Antihypertensive Activity in
Hypertensive Rats
Effects on the systolic blood pressure were determined
by intravenous injection of testing peptide into male
spontaneous hypertensive rats (SHR) (obtained form the
Animal Center of National Science Council, Taipei, Taiwan)
that were in an air-conditioned room (25 ± 1°C) having a
12:12 light-dark cycle (light on at 06:00). Food and water
were available ad libitum. All animal procedures were
performed according to the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health,
as well as the guidelines of the Animal Welfare Act. The
body weight of rats used was between 410 to 550 g and the
solution of testing peptide prepared at desired concentration
was injected at a ratio of 1 mL/kg of body weight. The
blood pressure was measured by tail-cuff method using the
MK-2000 blood pressure meter (Muromachi Kikai, Tokyo,
Japan) as described previously(10). Systolic blood pressure
was then calculated from four measurements of one animal
at the desired times.
The testing peptide was dissolved in physiological salt
solution for intravenous administration. After injection of
testing peptide into tail vein of SHR, blood pressures were
measured at the desired intervals (10, 20, 40, 60 and 90 min)
and the time before injection (0 min).
V. Data Analysis
Data are expressed as the mean ± SEM for the number
(n) of IC50 testing in each group indicated in the table and
gures. Repeated measures analysis of variance (ANOVA)
was used to analyze the changes of blood pressure and
other parameters. The Dunnett range post-hoc comparisons
were used to determine the source of signicant differences
where appropriate. A p < 0.05 was considered statistically
signicant.
RESULTS
I. Characterization of ACE Inhibitory Peptide by
Preincubation Method
A HPLC analysis of Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr
(0.02 mM) after the incubation with ACE (28 mU) showed
that this peptide was totally converted by ACE into Lys-Val-
Arg-Glu-Gly-Thr and Thr-Tyr (Figure 1B) as compared with
that of before the incubation with ACE, which shows Lys-
Val-Arg-Glu-Gly-Thr-Thr-Tyr only (Figure 1A). Thus, Lys-
Val-Arg-Glu-Gly-Thr-Thr-Tyr was digested totally by ACE
in this preincubation method. Then, the IC50 values of this
peptide were also determined before and after preincubation
with ACE. The inhibitory activity of Lys-Val-Arg-Glu-
Gly-Thr-Thr-Tyr was intensied about six times from IC50
= 102.8 μM before the incubation with ACE to IC50 = 17.2
μM after the incubation. However, preincubation with ACE
did not alter the inhibitory activity of Lys-Val-Arg-Glu-Gly-
Thr (Table 1).
II. Antihypertensive Activity of Testing Peptide In Vivo
The antihypertensive effects of Lys-Val-Arg-Glu-Gly-
Thr-Thr-Tyr, the original peptide, and Lys-Val-Arg-Glu-
Gly-Thr, the product (active form) after preincubation with
ACE, were determined in SHR. As shown in Figure 2, Lys-
Val-Arg-Glu-Gly-Thr-Thr-Tyr exerted a dose-dependent
(from 1 to 1000 pmol/kg, i.v.) antihypertensive activity by
an intravenous injection. Similar antihypertensive action
of Lys-Val-Arg-Glu-Gly-Thr was also obtained at the same
dose range (Figure 2). However, as shown in Figure 3,
the maximal decrease of systolic blood pressure (-10.3% =
-21.1 ± 4.1 mmHg) by Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr
Journal of Food and Drug Analysis, Vol. 14, No. 1, 2006
33
(1 nmol/kg, i.v.) was occurred 40 min after an intravenous
injection. But, maximal decrease of systolic blood pressure
(-11.6 % = -23.7 ± 2.3 mmHg) by Lys-Val-Arg-Glu-Gly-Thr
at same dose was observed 20 min after similar treatment.
Moreover, both at 10 and 100 pmol/kg (i.v.), Lys-Val-Arg-
Glu-Gly-Thr-Thr-Tyr decreased the systolic blood pressure
in SHR after 40 min of intravenous injection. Also, Lys-
Val-Arg-Glu-Gly-Thr produced the blood pressure lowering
action after 20 min of same treatments (data not shown).
DISCUSSION
ACE inhibitory peptides can be classified into three
groups, depending on their interaction with ACE(3,11,12).
The rst group is the true inhibitor, which inhibitory activity
is not altered by preincubation with ACE. The second
group comprises the substrates for ACE, which converts
substrate into inactive peptide, resulting in extensively
reduced activity of the peptide by incubation with ACE.
The third group is called pro-drug like inhibitory peptides.
They are also the substrates for ACE, but they are converted
by this enzyme into true inhibitors, resulting in an increase
of inhibitory activity after the preincubation with ACE.
According to the previous reports(3,5,8,13), only true inhibitor
and pro-drug like inhibitory peptide have the ability to
lower blood pressure. To distinguish true inhibitor from the
substrate, peptides were preincubated with ACE before the
measurement of ACE inhibitory activity. Basically, IC50
values of the true inhibitors are not affected by preincubation
with ACE, whereas substrates for ACE are changed by
preincubation with ACE. We found that Lys-Val-Arg-Glu-
Gly-Thr-Thr-Tyr is a pro-drug type ACE inhibitor because
preincubation with ACE of this peptide intensied the ACE
inhibitory activity about six times from IC50 value 102.8
to 17.2 μM (Table 1). This is also confirmed by HPLC
analysis of reaction mixture after preincubation showing that
ACE converts Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr to Lys-
Val-Arg-Glu-Gly-Thr (Figure 1). This result indicates that
Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr is hydrolyzed by ACE
into Lys-Val-Arg-Glu-Gly-Thr, the true ACE inhibitory
peptide because IC50 values for this peptide before and after
preincubation with ACE were found to be almost unchanged
(Table 1). The active peptide (Lys-Val-Arg-Glu-Gly-
Thr) showed an IC50 value of 7.6 μM that was lower than
that of 9 μM produced by Val-Pro-Pro(14) which exhibited
antihypertensive activity(7). However, the IC50 value of
Lys-Val-Arg-Glu-Gly-Thr is markedly higher than that of
captopril (22 nM), one of ACE inhibitors(3).
In order to assure of this hypothesis, peptides were
investigated in animals. Both of Lys-Val-Arg-Glu-Gly-
Thr-Thr-Tyr and Lys-Val-Arg-Glu-Gly-Thr caused an
antihypertensive activity after intravenous injection into
SHR. Similar dose-dependent antihypertensive activity after
an intravenous injection of two peptides from 1 to 1000
Tabl e 1. ACE inhibitory activity of the peptide derived from
ovotransferrin. Comparison of IC50 values before and after incubation
with ACE
Peptide IC50a (μM)
Before After
incubation incubation
with ACE with ACE
KVREGTTY 102.8 ± 11.9 17.2 ± 2.9
KVREGT 9.1 ± 1.8 7.6 ± 1.1
a
IC50: The concentration of each peptide required to inhibit 50% of
ACE activity. IC50 values were expressed as mean ± SEM.
Figure 1. Stability of KVREGTTY during the incubation with ACE.
RP-HPLC analysis of reaction mixtures before and after incubation
with ACE on a LiChroCART C18 column (4 mm I.D. × 250 mm).
Chromatograms (A) before preincubation and (B) after preincubation
with ACE. Peptide (0.02 mM) was incubated with 28 mU ACE (37°C,
3 hr).
0 4 8 12 16 20 24
0 4 8 12 16 20 24
Retention time (min)
Retention time (min)
(A)
(B)
0.6
0.3
0.0
0.6
0.4
0.2
0.0
-0.2
Absorbance at 214 nmAbsorbance at 214 nm
KVREGTTY
KVREGT
TY
Journal of Food and Drug Analysis, Vol. 14, No. 1, 2006
34
pmol/kg (i.v.) was observed (Figure 2). It seems reliable to
consider that the same peptide is responsible for the decrease
of systolic blood pressure after injection. Figure 3 showed
the time-course of systolic blood pressure change in SHR
induced by two peptides (1 nmol/kg, i.v.), Lys-Val-Arg-Glu-
Gly-Thr-Thr-Tyr and Lys-Val-Arg-Glu-Gly-Thr. Lys-Val-
Arg-Glu-Gly-Thr-Thr-Tyr showed the maximal decrease of
systolic blood pressure at 40 min after injection, while Lys-
Val-Arg-Glu-Gly-Thr produced the maximal effect at 20
min after injection (Figure 3). Moreover, both peptides at
10 or 100 pmol/kg (i.v.) administered into SHR produced a
similar time-course change of systolic blood pressure as that
of 1 nmol/ml/kg (i.v.) (data not shown). This 20 min delay
for the reduction of systolic blood pressure observed might
be considered as the time required for conversion of Lys-
Val-Arg-Glu-Gly-Thr-Thr-Tyr by ACE into the true inhibitor
of Lys-Val-Arg-Glu-Gly-Thr in SHR. Thus, a conversion
of Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr by ACE into the true
inhibitor of Lys-Val-Arg-Glu-Gly-Thr was identified in
vivo. Altogether, Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr can be
considered as a pro-drug type of ACE inhibitory peptide.
However, it should be noticed that the endogenous situation
is more complicated because other peptidases may also
participate in the degradation of peptides. A direct analysis
of the ACE activity in serum of SHR modied by the testing
peptide will be helpful. However, the employed method
for analysis of ACE activity was available for in vitro assay
only. Thus, more studies are needed to clarify the detailed
mechanism of this action in human subjects in the future.
In conclusion, the obtained results indicate that Lys-
Val-Arg-Glu-Gly-Thr-Thr-Tyr works as a pro-drug type ACE
inhibitory peptide because it was converted by hydrolysis
with ACE into Lys-Val-Arg-Glu-Gly-Thr, exhibiting a
11-fold augmentation in ACE inhibitory activity.
ACKNOWLEDGEMENTS
We appreciate Dairy Science Laboratory, Department
of Animal Product Science, Graduate School of Agriculture,
Hokkaido University (Sapporo, Japan) for technical support.
Thanks are also due to the kind help of colleagues in
Livestock Research Institute (Tainan, Taiwan, ROC).
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