Apolipoprotein A-I peptide models as probes to formulate potential inhibitors of the low-density lipoprotein oxidation

Article abstract of DOI:10.1002/psc.1391

Apolipoprotein A-I (apoA-I), which constitutes the principal protein component of high-density lipoprotein, is responsible for its major antiatherogenic functions. Aiming at contributing to the development of potent inhibitors of low-density lipoprotein (LDL) peptide models of helices 4,6 and 9,10 of apoA-I were designed and synthesized. Specific amino acid substitutions, resulting in transformation of the original helix class A and Y to G according to the Schiffer and Edmundson helical wheel representation, were introduced in order to validate the contribution of these modifications in the inhibitory activity of the synthesized peptide models against the LDL oxidation. The role of Met at positions 112 (helix 4) and 148 (helix 6) as oxidant scavenger was also investigated. The helical characteristics of all the peptide models were studied by CD in membrane-mimicking microenvironments and compared with the original helices.

Full text of DOI:10.1002/psc.1391

Research Article
Received: 25 January 2011
Revised: 24 March 2011
Accepted: 2 June 2011
Published online in Wiley Online Library: 22 September 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1391
Apolipoprotein A-I peptide models as probes
to formulate potential inhibitors of the
low-density lipoprotein oxidation
Maria I. Darvari,a Maria P. Petraki,b Constantinos Tellis,b
Konstantinos Harilogis,b Alexandros D. Tselepisb
and Maria Sakarellos-Daitsiotisb∗
Apolipoprotein A-I (apoA-I), which constitutes the principal protein component of high-density lipoprotein, is responsible for
its major antiatherogenic functions. Aiming at contributing to the development of potent inhibitors of low-density lipoprotein
(LDL) peptide models of helices 4,6 and 9,10 of apoA-I were designed and synthesized. Specific amino acid substitutions,
resulting in transformation of the original helix class A and Y to G according to the Schiffer and Edmundson helical wheel
representation, were introduced in order to validate the contribution of these modifications in the inhibitory activity of the
synthesized peptide models against the LDL oxidation. The role of Met at positions 112 (helix 4) and 148 (helix 6) as oxidant
scavenger was also investigated. The helical characteristics of all the peptide models were studied by CD in membrane-
c
mimicking microenvironments and compared with the original helices. Copyright ꢀ 2011 European Peptide Society and John
Wiley & Sons, Ltd.
Keywords: apolipoprotein A-I helical peptide models; inhibitors of the LDL oxidation; HDL; CD of apoA-I helices
Introduction
ofhelix4and148ofhelix6[8].ApoA-Iplaysalsoacentralregulatory
role in the cholesterol efflux via ABCA1 considering to this action
helices 9 and 10 spanning the sequence 209–229 [9–11].
Numerous studies have shown that the high levels of high-density
lipoprotein (HDL) and its most abundant protein constituent
apolipoprotein A-I (apoA-I) can reduce the risk of atherosclerosis,
the leading cause of death in industrialized countries. It is well
documented now that apoA-I, which constitutes the principal
protein component (70%) of HDL, is responsible for the major
functions of HDL as lipid binding and solubilization, cholesterol
removal from peripheral cells, activation of lecithin-cholesterol
acyltransferase (LCAT) and its antioxidant and anti-inflammatory
effects. Several studies have also shown that the intrinsic flexibility
of apoA-I allows its structure to acquire different conformations in
different lipid environments [1–6].
BasedonX-ray crystallographyandcomputermodelling, apoA-I
encoded amino acids form 22- and 11-tandem repeats, which are
organized in ten amphipathic α-helices, eight α-helical segments
of 22 amino acids and two 11-mer repeats that are frequently
separated by proline residues.
Amphipathic α-helices of apoA-I are assigned to the class A,
Y and G depending on the distribution of the positively and
negatively charged residues on the polar face [12–17]. The most
distinctive feature of the class A helix is the unique clustering of
positively charged residues at the polar–non-polar interface and
the negatively charged residues at the centre of the polar face.
In class Y, positively charged residues are located at the interface
and the centre of the polar face, whereas negative-charged
residues occupy the rest of the polar face. Class G helices have
a random distribution of negative and positive charges around
the perimeter of the polar face, in contrast to the highly typical
clustering seen in class A [18–25].
Aiming at contributing to the development of potent athero-
protective agents peptide models of helices 4, 6 and 9, 10 were
designed and synthesized: specific amino acid substitutions, re-
sulting in transformation of the original helix class A and Y to
G according to the Schiffer and Edmundson [26] helical wheel
representation, were introduced in order to validate the con-
tribution of these modifications in the antioxidant functions of
the peptide models. The role of Met at positions 112 (helix 4)
TheN-terminalhelix2andtheC-terminalhelix10arerecognized
as important for the initial association with lipids. In the central
domain, helix 4 and, to a lesser extent, helix 5 are also very
important for lipid binding and the formation of mature HDL,
whereas helices 6 and 7 contribute little [4].
A key metabolic role of apoA-I is its ability to activate
LCAT, an enzyme which transesterifies the sn-2 fatty acid from
phospatidylcholine to cholesterol to yield cholesteryl esters [7].
This role has been devoted to helix 6 of apoA-I, which includes
the amino acids sequence 143–164 of apoA-I molecule. Moreover,
some of the antioxidant properties of apoA-I have been attributed
tospecificaminoacidsinthatmoleculesuchasMetatpositions112
∗
Correspondence to: Maria Sakarellos-Daitsiotis, Department of Chemistry,
University of Ioannina, 45110 Ioannina, Greece. E-mail: msakarel@uoi.gr
a
Department of Medicine, Division of Cardiology, New York University School of
Medicine, New York, NY, USA
b
Department of Chemistry, University of Ioannina, Ioannina, Greece
c
J. Pept. Sci. 2011; 17: 720–725
Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.

PEPTIDE MODELS OF APOLIPOPROTEIN A-I
Table 1. Synthesized peptide models of apoA-I (1–9)
Peptide models
Helix-class
Position in apoA-I
Sequence
1
2
Helix 4-class Y
Helix 4-class G
104–117
Ac-FQKKWQEEMELYRQ-NH2
Ac-FQKKWQEEAELYRQ-NH2
104–117
Met112 →Ala
3
4
Helix 6-class A
Helix 6-class A
147–159
Ac-EMRDRARAHVDAL-NH2
Ac-EARDRARAHVDAL-NH2
147–159
Met148 →Ala
5
Helix 6-Class G
147–159
Ac-EMNVaOrnRARAAVDAL-NH2
Arg149 →Nva
Asp150 →Orn His155 →Ala
152–164
6
7
Helix 6-class A
Helix 6-class G
Ac-ARAHVDALRTHLA-NH2
Ac-ARAHVDALRKHLA-NH2
152–164
Thr161 →Lys
8
9
Helix 9,10-class Y
Helix 9,10-class G
209–229
Ac-PALEDLRQGLLPVLESFKVSF-NH2
Ac-PALEOrnLRQGLLPVLEKFKVSF-NH2
209–229
Asp213 →Orn
Ser224 →Lys
and 148 (helix 6) as oxidant scavenger was also investigated. All
peptide models (Table 1, 1–9) were tested for their inhibition to
the low-density lipoprotein (LDL) oxidation and their conforma-
tional characteristics were studied by CD in membrane-mimicking
microenvironments.
Analytical RP-HPLC
Analytical HPLC of the synthesized compounds (1–9) was
performed using a Supelco Discovery (Sigma Aldrich, USA), Waters
616 (USA) (Sigma Aldrich, USA), Waters 616 (USA) C18 (25 cm ×
4.6 mm, 5 µm) reverse-phase column with a Waters instrument
equipped with a Waters 616 pump and a Waters 2487 (USA) dual
λ absorbance detector. Eluent A was 0.1% TFA in water and eluent
B was 0.1% TFA in acetonitrile. A linear gradient was used from
10 to 70% acetonitrile in 0.1% TFA at a flow rate of 1 ml/min
(1/1/0.05 v/v/v).
Materials and Methods
Peptide Synthesis
The synthesis of the peptides was carried out by the stepwise
solid-phase procedure SPPS [27,28] on a Rink Amide AM resin
(GLBIOCHEMShangHai,China)(0.67 mmol/gresin)usingtheFmoc
methodology. Lys was introduced as Fmoc-Lys-(Boc)-OH (Boc:
t-Butoxycarbonyl), glutamic acid as Fmoc-Glu(OtBu)-OH (OtBu: t-
Butoxy), glutamine as Fmoc-Gln(Trt)-OH (Trt: Trityl), tryptophan as
Fmoc-Trp(Boc)-OH, arginine as Fmoc-Arg(Pbf)-OH (Pbf: 2,2,4,6,7-
pentamethyl-dihydrobenzofurane-5-sulfonyl), tyrosine as Fmoc-
Tyr(tBu)-OH (tBu: tert-butyl), serine as Fmoc-Ser(tBu)-OH, histidine
asFmoc-His(Boc)-OH, ThrasFmoc-Thr(tBu)-OH, ornithineasFmoc-
Orn(Boc)-OH and aspartic acid as Fmoc-Asp(OtBu)-OH. Fmoc
groups were removed using 20% piperidine in DMF. Coupling
of each Fmoc-amino acid (3 mol equiv.) was performed in the
presence of HBTU/HOBt/DIEA (2.9/3/6 molar ratio). DMF was
distilled over ninhydrin to remove traces of amines. Completion
of the couplings was ensured by the ninhydrin Kaiser test.
Acetylation was performed using a 30 : 1 ratio of acetic anhydride
(Ac2O)/NH2 groups in pyridine. Peptide models incorporating Met
were cleaved from the resin by treatment with TFA/TIS/DMS/H2O
(92.5/2.5/2.5/2.5) and the rest of them with the TFA/TIS/H2O
(95/2.5/2.5) treatment. The resin was removed by filtration, the
filtrateswereevaporatedunderreducedpressureandtheproducts
were precipitated with cold diethyl ether. Yields range from 80
to 90%. The crude peptides were purified by semi-preparative
reverse-phase HPLC on a C18 column. Appropriate programmed
gradients were applied using eluants A (H2O/0.1% TFA) and B
(CH3CN/0.1% TFA). The purity of the peptides was checked by
analytical HPLC and the correct molecular masses were confirmed
by ESI-MS. Molecular weights (M+) of the peptides are presented
in Table 2.
Semi-preparative RP-HPLC
RP-HPLC purification was carried out on a SHIMADZU semi-
preparative instrument equipped with a SHIMADZU LC-10AD
(Shimadzu Corporation, Japan) VP pump and a SHIMADZU SPD-
10A VP UV-VIS (Shimadzu Corporation, Japan) VP UV-VIS detector,
using a Supelco Discovery C18 (25 cm × 10 mm, 5 µm) column,
with the same linear gradient as for analytical RP-HPLC at a flow
rate of 4.7 ml/min. Detection was carried out at λ = 214 nm.
Mass spectroscopy
Positive ion ESI-MS analyses of synthesized compounds 1–9
were performed on a Micromass Platform (Canton, MA 02021
USA) LC-MS. Capillary and cone voltages were set to 3 kV
and 35–75 V, respectively. Samples were dissolved in wa-
ter/acetonitrile/trifluoroacetic acid; molecular weights calculated
and found are given in Table 2.
Circular Dichroism
CD spectra were recorded at 25 ◦C on a Jasco J-815 (Jasco Corpo-
ration, Japan) spectropolarimeter equipped with a thermoelectric
temperature controller. Spectra were obtained using a quartz
cell of 1-mm path length and the concentration of the tested
compounds was 10−4 M. Experiments were performed in PBS pH
7.4, SDS 8 mM, C14PC 5 mM (dimyristoyl phosphatidylcholine) and
TFE/H20 mixtures (50 : 50 v/v). Spectra were obtained with a 1 nm
bandwidth, a scan speed of 50 nm/min and a response of 1 s.
c
J. Pept. Sci. 2011; 17: 720–725 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

DARVARI ET AL.
Table 2. Parameters of the synthesis, purification and characterization of peptide models 1–9
RP-HPLC (gradient
Peptide
1
Yield (%)
85
elution), A : B
tR (retention time) (min)
ESI-MS
90 : 10
30 : 70
95 : 5
14
Calculated [M+H]+: 1984.28
Found [M+H]+: 1985.74
Calculated [M+H]+: 1924.15
Found [M+H]+: 1924.49
Calculated [M+H]+: 1580.78
Found [M+H]+: 1580.95
Calculated [M+H]+: 1520.67
Found [M+H]+: 1520.90
Calculated [M+H]+: 1456.72
Found [M+H]+: 1457.32
Calculated [M+H]+: 1471.70
Found [M+H]+: 1472.05
Calculated [M+H]+: 1498.80
Found [M+H]+: 1499.63
Calculated [M+H]+: 2399.82
Found [M+H]+: 2400.72
Calculated [M+H]+: 2439.96
Found [M+H]+: 2440.72
2
3
4
5
6
7
8
9
79
80
80
87
66
87
74
65
12.5
11
30 : 70
95 : 5
30 : 70
95 : 5
14.5
19
30 : 70
90 : 10
40 : 60
90 : 10
50 : 50
90 : 10
50 : 50
70 : 30
20 : 80
90 : 10
20 : 80
21
11
12
16
A, H2O/0.1% TFA; B, CH3CN/0.1% TFA.
The signal-to-noise ratio was improved by accumulating three
scans. All CD spectra are reported in terms of mean residue molar
ellipticity (θ) in degree cm2/dmol). The percentage helical content
was estimated on the basis of the [ꢀ]222 nm values, in different
environments, as described by Chen et al. [29].
constituents of HDL, which plays a critical role to the HDL func-
tions, remains a challenging goal for the development of potent
atheroprotective agents.
Association of apoA-I with lipids, a crucial functional step of
apoA-I, do not appear to be simply related to the hydrophobicity
of the protein α helices but also to the distribution of charged
residuesalongthehelix,whichisdirectlyimplicatedtothedifferent
classes of α helices. Peptide models obtained by the conversion
of the most abundant classes A and Y to class G were designed,
synthesized and studied in order to investigate the effect of the
random distribution of the positive and negative charges on the
polar phase of the helices in their conformational and biological
properties.
Substitution of Arg149 →Nva, Asp150 →Orn and His155 →Ala
in peptide model 3, helix 6 class A, resulted in the formation of a
class G peptide model (5). Substitution of Thr161 for Lys in helix
6 (peptide model 6) converted also class A to class G peptide
model (7). Replacement of Asp213 →Orn and Ser224 →Lys in helix
9,10 class Y (peptide model 8) created a class G peptide model
(9) (Table 1).
Among the four Met residues comprised within the amino acid
sequence of apoA-I, two of them at positions 112 and 148 are likely
to play an important role as oxidant scavengers [33]. Met112 and
Met148 in helix 4 and 6 respectively were substituted by Ala and
the antioxidant effects of the resulting peptide models (2,4) were
compared with the initial helices (1,3) (Table1).
Helices 4,6 and 9,10, as well as their substituted models were
synthesized according to the solid-phase peptide method in good
yields and high purity. Parameters of the synthesis, purification
and characterization of peptide models 1–9 are given in Table 2.
The CD spectra of peptide models 1–9 in SDS, at the critical
micelle concentration 8 mM, and in C14PC 5 mM exhibited a
positive band at 192 nm and two negative bands at 205 and
222 nm typical of helical structure (Figures1 and 2). Their helical
content, estimated on the basis of the [ꢀ]222 value, ranged
from 32 (6,8) to 83% (5) in SDS and from 29 (3,4) to 62% (5) in
LDL Isolation and Oxidation
LDL (d = 1.019–1.063 g/ml) was isolated from freshly prepared
humanplasmasupplementedwith0.01%EDTA(w/v)and5 mg/ml
gentamicin sulfate Garamycin, Schering-Plough, (NY, USA) by
sequential centrifugation in a Beckman L7-65 ultracentrifuge at
40 000 rpm, 14 ◦C with a Type NVT 65 rotor [30]. LDL was filter
sterilized (0.22 mM, Millipore, Billerica, MA, USA) and stored in
the dark at 4 ◦C under nitrogen for up to 2 weeks. LDL protein
was determined by the bicinchoninic acid method. Purity of the
LDL preparation was assessed by agarose gel electrophoresis
Hydragel Lipo ad Lp(a) Kit (Sebia, Norcross, GA 30093, USA).
Before oxidation, LDL was extensively dialysed (to remove EDTA)
against two changes of a 200-fold volume of 10 mM PBS for 24 h
at 4 ◦C in the dark. LDL (100 µg protein/ml) was incubated in
the presence of copper sulfate (5 µM final concentration) [31]. In
selected experiments LDL (100 µg protein/ml) was oxidized with
met-myoglobin/H2O2 (10 and 50 mM, respectively) in the presence
of diethylenetriaminepentaacetic acid (DTPA) (0.1 mM) [17].
In all oxidation studies, peptides were dissolved in PBS, pH 7.4,
and used at final concentration ranging from 40 to 424 µM. The
kinetics of oxidation were determined by monitoring the increase
in absorbance at 234 nm every 10 min for 6 h. The lag time of
peptide oxidation was calculated as previously described [32].
Results and Discussion
One of the mechanisms by which HDL might be antiatherogenic
is by protecting LDL from oxidation and inhibiting the proathero-
genic effect of oxLDL in vitro and in vivo. ApoA-I, one of the main
c
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PEPTIDE MODELS OF APOLIPOPROTEIN A-I
Table 3. Effect of the apoA-I peptide models 1–9 on Cu2+-induced
LDL oxidation
60
45
30
15
0
Peptide model 5
Peptide model 6
Peptide model 9
Reduction of the Reduction of
Peptide Concentration Lag time
oxidation
rate (%)
total dienes
(%)
model
(µM)
(min)
–
1
2
3
4
5
6
7
8
9
0
424
424
318
318
40
60 10
86
–
–
50
22
8.7
8
67
40
65
10
100
100
45
27
40
70
160
100
–
100
100
2
-15
-30
53
–
424
424
424
110
90
5.4
8
120
wavelength (nm)
Figure 1. CD spectra of peptide models 5, 6 and 9 in SDS 8 mM.
and decreased the maximal rate of conjugated diene formation
(rate of oxidation), as well as the total amount of dienes formed
during oxidation, in a dose-dependent manner (Table 3).
Peptide model 3 corresponding to helix 6 (147–159) class A
increased the lag time of LDL oxidation (from 60 to 160 min) and
decreased the rate of oxidation to 64% and the diene formation
to 8.7% in a dose-dependent manner (range of concentrations
53–318 µM) (Figure 3(A), Table 3). Peptide model 5, obtained from
the conversion of class A to class G, exhibited a significantly
more potent inhibitory effect compared to 3 as its threshold
concentration was 40 µM versus 318 µM of 3 (Table 3). In fact the
random distribution of positive and negative charges on the polar
face of class G, in contrast to the typical clustering of class A, as well
as the relatively high percentage of helical conformation, defined
by CD, seems to favour the inhibition of the peptide model 5 on
the LDL oxidation (Figure 3(B)).
45
30
15
0
Peptide model 5
Peptide model 6
Peptide model 9
-15
-30
The inhibitory effect of peptide models on Cu2+-induced LDL
oxidation was rather potent as the peptide model 6 corresponding
to helix 6 (152–164) class A (6) inhibited oxidation completely
at the concentration 0.53 µM. On the contrary the inhibitory
properties of model 7 class G, resulting from model 6 after the
substitution of Thr161 for Lys, were relatively weak. The rate of
oxidation decreased to 45% and the diene formation to 2% (range
of concentrations 0.53–424 µM). It is likely that replacement of Thr
from Lys modified the hydrophobicity of model 7 and diminished
its activity (Table 3).
Peptide model 8 spanning the sequence 209–219 of helices
9,10 class Y reduced the rate of oxidation to 27% and the diene
formation to 5.4% (concentrations from 53 to 424 µM). Peptide
model 9 class G obtained by substitution of Asp213 →Orn and
Ser224 →Lys showed 40% depletion of the LDL oxidation rate and
8% reduction of the diene formation (concentrations ranged from
53 to 424 µM). These findings point out that the transformation of
peptidemodel8fromclassYtoclassG, aswellastheenhancement
of helix, stimulates the inhibitory activity of the resulting peptide
model 9 (Figure 4 and Table 3).
Oxidation of LDL in the presence of peptide model 1, which
incorporatesMetatposition112, wasinhibitedconsiderablyasthe
decrease of the oxidation rate and the diene formation reached
67 and 50%, respectively (concentrations from 53 to 424 µM). On
the contrary, substitution of Met112 by Ala (2) reduced the rate
of oxidation and the diene formation to 40 and 22% respectively
confirming the role of Met as an oxidant scavenger. Peptide
models 3 and 4 showed comparable activities (∼65% reduction
wavelength (nm)
Figure 2. CD spectra of peptide models 5, 6 and 9 in C14PC 5 mM.
C14PC, whereas in phosphate-buffered saline (PBS) they adopt
random structure. These findings point out that the synthesized
modelsexperiencehelicalconformationsinmembrane-mimicking
microenvironments. The helical conformation of all peptide
models was also preserved in TFA/H2O mixtures (50 : 50 v/v),
ranged from 22 to 70%, as TFA is a solvent that favours the
helix.
It deserves to be mentioned that transformation of class A
helix 6 (147–159) (3) to class G (5) duplicated the helical content
in C14PC (from 30 to 60%) and enhanced the helix from 50 to
83% in SDS. Significant increase of the helical features of class
A helix 6 (152–164) (6) and class Y helix 9,10 (209–229) (8) was
also observed after their alterations to class G (7,9). One may
assume that transformation of class A and Y to G increased the
helical characteristics of the helices, which is attributed to a more
favourable distribution of the charges on the polar phase of G
compared to A and Y.
The inhibitory activity of the synthesized peptide models (1–9)
wasevaluatedonthebasisoftheirabilitytoincreasetheresistance
of isolated LDL to copper-induced oxidation in vitro. The kinetics
of the oxidation were determined by monitoring the increase of
the absorbance at 234 nm in various peptide concentrations. All
the tested peptides (1–9) increased the lag time of LDL oxidation
c
J. Pept. Sci. 2011; 17: 720–725 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

DARVARI ET AL.
1.20
1.00
0.80
0.60
0.40
0.20
0.00
(A) 1.6
1.4
None
53 µM
106 µM
212 µM
318 µM
1.2
Control
1.0
Peptide-1
Peptide-3
Peptide-4
Peptide-5
Peptide-6
Peptide-7
Peptide-9
0.8
0.6
0.4
0.2
0.0
0
50
100
150
200
0
50
100
150
200
250
300
350
Time (min)
Time (min)
Figure 5. Representative curves of LDL oxidation with met-myoglobin/
H2O2/DTPA in the presence of peptide models at 424 µM.
(B) 2.0
1.8
None
212 µM
318 µM
424 µM
53 µM
1.6
1.4
system that lacks free iron ions as any possible contaminating iron
is chelated with DTPA [17]. The interaction of met-myoglobin with
H2O2 leads to the formation of ferryl myoglobin-free radicals that
are able to oxidize vulnerable targets, like unsaturated lipids [34].
The amount of total dienes formed during LDL oxidation reached a
maximumof215 54 nmol/mgproteinat80 10 minofoxidation.
Similar oxidation curves were obtained in the presence of all
peptides tested (Figure 5), indicating that peptide models failed
to inhibit LDL oxidation performed in the absence of transition
metals. These results suggest that the antioxidant effect of peptide
models investigated in this study is at least partially attributed to
transition metal chelation. However, the enhanced antioxidant
activity of peptide models 5 and 6, from tenfold to eightfold
compared with the rest, indicates their effectiveness in protecting
LDL from oxidation.
106 µM
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
50
100
150
200
250
Time (min)
Figure 3. Sigmoid oxidation curves illustrating the dose-dependent
inhibition of LDL oxidation induced by the peptide model 3 (A) and 9
(B).
1.6
1.4
1.2
1.0
Conclusions
Our study points out that the conversion of the apoA-I helices
from one class to another might affect their biological properties
as the charge distribution on the polar phase of the helices is
related to their affinities towards lipoproteins. Thus, alteration
of class A and Y peptide models (3 and 8) to class G (5 and
9) augmented their antioxidant activities. In particular peptide
model 5, derived from the conversion of model 3 (class A) to
G, inhibited the LDL oxidation at a relatively low concentration
40 µM. However, substitution of Thr for Lys in peptide model 6
(class A) abolished the activity of model 7 (class G) indicating how
perceptive is the balance of the charges on the polar phase of the
helix. The contribution of Met at positions 112 and 148, as oxidant
scavengers, was also confirmed as their replacement reduced the
antioxidant properties of models 2 and 4. It is concluded that
modifications of the apoA-I helices might lead to the formulation
of potential atheroprotective peptide model candidates.
None
0.8
Peptide model 3
0.6
Peptide model 4
0.4
Peptide model 5
0.2
0.0
0
50
100
150
200
250
Time (min)
Figure 4. Sigmoid oxidation curves illustrating the dose-dependent
inhibition of LDL oxidation induced by the peptide models 3, 4 and 5
at 424 µMU.
of the oxidation rate and ∼8% reduction of the diene formation)
indicating that the replacement of Met148 (3) by Ala (4) was not
crucial (Table 3).
According to our previously published results, purified apoA-
I, almost completely inhibits Cu2+-induced LDL oxidation at a
concentration of 321 µM [17]. In the present study, the peptide
models5and6completelyinhibitLDLoxidationataconcentration
of 40 and 53 µM, respectively, thus these peptides are sixfold to
eightfoldmorepotentcomparedwithapoA-I.Totestthepossibility
of these peptides acting as transition metal chelators, through
their polar side chains, we studied their effect on LDL oxidation
performed in the presence of met-myoglobin/H2O2 and DTPA a
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PEPTIDE MODELS OF APOLIPOPROTEIN A-I
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