Measurement of Copper-Binding Sites on
Low Density Lipoprotein
Alexander Roland, Rebecca A. Patterson, David S. Leake
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Abstract—Copper is often used to oxidize low density lipoprotein (LDL) in experiments in vitro and is a candidate for
oxidizing LDL in atherosclerotic lesions. The binding of copper ions to LDL is usually thought to be a prerequisite for
LDL oxidation by copper, although estimates of LDL copper binding vary widely. We have developed and validated
an equilibrium dialysis assay in a MOPS-buffered system to measure copper binding to LDL and have found 38.6⫾0.7
(mean⫾SEM, n⫽25) copper binding sites on LDL. The binding was saturated at a copper concentration of 10 ␮mol/L
at LDL concentrations of up to 1 mg protein/mL. Copper-binding capacity increased progressively and markedly when
LDL was oxidized to increasing extents. Chemical modification of histidyl and lysyl residues on apolipoprotein B-100
reduced the number of binding sites by 56% and 23%, respectively. As an example of the potential of this method to
assess the effects of antioxidants on copper binding to LDL, we have shown that the flavonoids myricetin, quercetin,
and catechin (but not epicatechin, kaempferol, or morin), at concentrations equimolar to the copper present (10 ␮mol/L),
significantly decreased copper binding to LDL by 82%, 56%, and 20%, respectively. (Arterioscler Thromb Vasc Biol.
2001;21:594-602.)
Key Words: low density lipoproteins 䡲 atherosclerosis 䡲 copper 䡲 flavonoids 䡲 quercetin
T
pro-oxidant enzyme myeloperoxidase can be found in early
as well as in advanced atherosclerotic lesions.20 These mechanisms and others may play a role in atherosclerosis, although
their relative importance may differ at different stages of the
disease.
For in vitro studies of oxidized LDL, oxidation by copper
is frequently used and produces LDL with characteristics
similar to LDL oxidized by cells.1 Copper is more potent than
iron in its ability to oxidize LDL in vitro.21 The oxidation of
LDL by copper is likely to require the binding of copper ions
to the lipoprotein particle. This binding occurs, at least in
part, to histidine-containing sites on apoB-100, the protein
moiety of LDL.22,23 The number of binding sites reported
varies widely over 2 orders of magnitude22–33 (Table).
A number of antioxidants may act in part by inhibiting the
binding of copper (or iron) to LDL. Flavonoids provide a
potentially important example of antioxidants that may function in this way. Flavonoids are potent inhibitors of LDL
oxidation by transition metal ions and cells,34 –36 and dietary
intake of these compounds has been inversely correlated with
cardiovascular disease in several, but not all, epidemiological
studies.37– 43 Most biochemical studies have focused on the in
vitro free radical–scavenging activity of flavonoids, but a
number of flavonoids are capable of forming complexes with
transition metal ions.44 – 47
In the present study, we report an equilibrium dialysis assay
that has allowed us to determine the number of binding sites for
copper on LDL, and we demonstrate its value in assessing the
he oxidation of LDL is widely believed to be an
important event in the pathogenesis of atherosclerosis.1,2
Oxidized LDL may be endocytosed in an uncontrolled
manner by macrophages, resulting in the generation of
cholesterol-laden foam cells, which characterize atherosclerotic lesions.1,2 In addition, oxidized LDL is chemotactic for
leukocytes,3 is cytotoxic,4 can induce smooth muscle cell
proliferation,5 and has many other potentially atherogenic
effects.
The presence of active transition metal ions is usually a
prerequisite for LDL oxidation by cells, at least in vitro,6 –10
and LDL can also be oxidized by transition metal ions in the
absence of cells.6 – 8 The mechanisms of LDL oxidation in
atherosclerotic lesions are unclear, but transition metal ions
may be involved.11 Catalytically active copper and iron have
been reported to be present in atherosclerotic lesions,12–14 and
the copper-carrying protein of plasma, ceruloplasmin, can
catalyze LDL oxidation.15,16 At pH 7.4, only the single most
loosely held copper ion on ceruloplasmin appears to be
required to catalyze LDL oxidation,15 although the other
copper ions on ceruloplasmin may be important at acidic
pH.16,17 In addition, there appears to be a trend toward
increased levels of o- and m-tyrosine in human advanced
atherosclerotic lesions, which may be indicative of the
presence of redox-active copper.18 However, high levels of
3-nitrotyrosine, indicative of the presence of the oxidizing
agent peroxynitrite, can also be found in LDL isolated from
atherosclerotic lesions.19 Evidence of the importance of the
Received September 13, 2000; revision accepted December 19, 2000.
From the Cell and Molecular Biology Research Division, School of Animal and Microbial Sciences, The University of Reading, Reading, Berkshire, UK.
Correspondence to Alexander Roland, Cell and Molecular Biology Research Division, School of Animal and Microbial Sciences, The University of
Reading, Whiteknights, PO Box 228, Reading, Berkshire, RG6 6AJ, UK. E-mail [email protected]
© 2001 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
594
Roland et al
Copper Binding to LDL
595
Previously Reported Numbers of Binding Sites for Copper on LDL
Highest Ratio
of Copper/LDL
Examined
Method Used
Sephadex gel filtration
Buffer Used
Expected
Maximum Binding
in Used Buffer, n*
Copper Ions
Bound per LDL
Particle, n
Reference
9
NaCl (pH 7.4)
36
2†
24
Incubation followed by dialysis in
absence of copper, with or without
Sephadex gel filtration
34
NaCl (pH 7.4)
36
6–8†
26
Sephadex gel filtration
13
Phosphate (concentration
not stated)
12
1
30
100
Tris (10 mmol/L)
9
⬎50
25
Ultrafiltration to dryness I
50
HEPES (100 mmol/L)
27
25–28
22
Ultrafiltration I
82
Phosphate (10 mmol/L)
12
17
23
Ultrafiltration followed by LDL
proteolysis
10
Tris (5 mmol/L)
9
3
27
2
Polarography
Ultrafiltration II
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Phosphate (50 mmol/L)
12
1
28
Ultrafiltration to dryness II
205
HEPES (100 mmol/L)
27
64
33
Precipitation
122‡
Phosphate (concentration
not stated)
12
96
31
8
Phosphate (10 mmol/L)
followed by Tris (5 mmol/L)
9
7
29
Incubation followed by dialysis in the
absence of copper
Equilibrium dialysis
10§
Oxidation kinetics
220
HEPES (20 mmol/L)
27
5†
23
Phosphate (concentration
not stated)
12
17
32
The studies are arranged by method.
*Number of copper ions bound per LDL particle that we found at equilibrium in buffer used in the study.
†Values are calculated from authors’ data.
‡In 1 case, LDL concentration was given as mg total lipid/dL; we have estimated the concentration of the LDL as mol/L on the basis of an LDL particle with a
relative molecular mass of 2.5 million (as assumed by the authors) containing 78% lipid.
§Note that low copper concentrations are not necessarily limiting for copper binding to LDL when equilibrium dialysis is used, inasmuch as there is a large excess
of copper compared with LDL in this system because the LDL is confined to a small fraction of the total dialysis volume.
ability of antioxidants for LDL to inhibit the binding of copper
to these lipoproteins by the use of flavonoids.
Methods
LDL Isolation and Oxidation
LDL (density 1.019 to 1.063 g/mL) was isolated from the blood of
healthy volunteers by sequential density ultracentrifugation at 4°C in
KBr solutions, as described elsewhere.48 Oxidation was carried out to
defined extents at 37°C in Dulbecco’s PBS as previously described.49
Briefly, LDL (100 ␮g protein/mL) was incubated with CuSO4 (Fisher
Scientific UK) at a concentration 5 ␮mol/L above that of the EDTA
carried over from the LDL preparation. Conjugated diene formation was
monitored at 234 nm. Oxidation was stopped by the addition of EDTA
(1 mmol/L). Mildly oxidized LDL was defined as LDL that had
demonstrated an absorbance increase of 0.2, moderately oxidized LDL
was defined as LDL in which peak absorbance had been reached (after
⬇3 hours), and highly oxidized LDL was defined as LDL whose
oxidation had been terminated after 24 hours. The density of each
sample was adjusted to 1.2 g/mL with solid KBr, in the presence of
washed Chelex-100 chelating resin (Sigma). Chelex was prewashed in
distilled water to remove any contaminating antioxidant activity associated with the beads.50 After centrifugation at 250g for 10 minutes to
sediment the Chelex, LDL was ultracentrifuged at 150 000g for 18
hours at 4°C. The oxidized LDLs were then dialyzed at 4°C against
PBS, pH 7.4, consisting of NaCl (140 mmol/L), Na2HPO4 (8.1 mmol/L),
NaH2PO4 (1.9 mmol/L), and EDTA (100 ␮mol/L), before filter sterilization. LDL was stored in darkness under argon at 4°C and was used
within 4 weeks. Ethics committee approval was obtained for the
isolation of LDL at the University of Reading, and blood donors gave
their informed consent.
Copper Binding to LDL
Buffers were prepared at 4°C and pretreated with washed Chelex100 (1 g/L) to remove traces of transition metals. LDL was
extensively dialyzed in dialysis tubing (10-mm flat width) with a
molecular weight cutoff of 12 to 14 kDa (Medicell International Ltd)
against the phosphate buffer described above but without EDTA for
at least 12 hours at 4°C, with 3 buffer changes to remove EDTA. A
further buffer exchange was then carried out by dialyzing at 4°C against
a MOPS buffer, pH 7.4, consisting of NaCl (150 mmol/L), MOPS
(10 mmol/L), and butylated hydroxytoluene (BHT, 20 ␮mol/L), pH 7.4
(MOPS and BHT were obtained from Sigma). The LDL samples were
then diluted to 1 mg LDL protein/mL by using MOPS buffer containing
CuSO4 (10 ␮mol/L added from a 10-mmol/L stock solution in water),
with or without flavonoids (10 ␮mol/L), and 1 mL was loaded into
Medicell dialysis tubing or 15-kDa cutoff Spectra/Por 2.1 high-speed
dialysis tubing (Spectrum). Copper binding to LDL was carried out in
500 mL MOPS buffer containing copper and flavonoids at 4°C, pH 7.4,
until saturation of binding was achieved (24 hours with Medicell tubing,
10 hours with Spectra/Por 2.1 tubing). Gentle stirring of buffer solutions
was carried out at each dialysis stage to ensure mixing without inducing
LDL aggregation. After dialysis, LDL protein was measured by use of
a modified Lowry assay,51 after the LDL was diluted 10-fold in water.
An equivalent volume of MOPS buffer was included in the BSA
standards, because MOPS increased the absorbances obtained in the
protein assay. For experiments with other buffers, the above procedure
was carried out exactly as described above but substituting MOPS with
10 mmol/L of one of the following buffers: MES, HEPES, phosphate,
or Tris. NaCl (0.15 mol/L) of “pH 7.4” was also tested. When alternative
buffers were tested, these were also included in the BSA standards of the
protein assay.
Quantification of Copper
Copper ions were measured spectrophotometrically by use of the
indicator molecule bathocuproinedisulfonic acid (BC, Sigma), the
Cu(I) complex of which absorbs strongly at 480 nm.52,53 Triplicate
samples of LDL were diluted 4-fold in MOPS buffer; control
samples without LDL were obtained from the bulk dialysis solution
596
Arterioscler Thromb Vasc Biol.
April 2001
and from control dialysis bags containing only copper and dialysis
buffer to confirm that the “free” copper concentrations were equal on
both sides of the dialysis membrane. A 0.6-mL volume of each
diluted sample was added to 50 ␮L BC stock solution in water (to
give a final concentration of 400 ␮mol/L BC), together with 50 ␮L
ascorbate (sodium salt, Sigma) stock solution freshly prepared in
water (to give a final concentration of 1 mmol/L ascorbic acid). The
mixture was incubated at room temperature for 5 minutes, during
which any Cu(II) was reduced to Cu(I), resulting in BC-Cu(I)
complex formation. A further 0.6-mL sample of each diluted LDL
solution was incubated with EDTA (1 mmol/L added in a 100-␮L
volume of stock EDTA in water), for the reason explained in Results.
Absorbances were measured at 480 nm, and the values obtained in
the presence of EDTA were deducted from the values obtained in the
presence of BC/ascorbic acid (the former values were typically 10%
to 20% of the latter). Copper levels were determined by comparison
with a CuSO4 standard plot up to 100 ␮mol/L.
Calculation of Copper Ions Bound
per LDL Particle
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To determine the number of copper ions associated with each
lipoprotein particle, LDL concentrations were converted to micromolar units (a value of 513 kDa was used for the molecular mass of
apoB-100). After deducting the free copper concentration (see
above) from the total concentration of copper associated with the
LDL, the concentration of LDL-associated copper was divided by
the concentration of LDL to give a ratio of copper ions bound per
LDL particle.
We examined whether the presence of LDL interfered with the
standard plot for the copper assay. Standard plots (0 to 100 ␮mol/L
copper) in the presence of LDL (250 ␮g protein/mL before addition
of EDTA or BC and ascorbate), obtained by deducting EDTAcorrected absorbances from those in the presence of BC and
ascorbate, revealed that such plots were indistinguishable from those
obtained in the absence of LDL at copper concentrations ⬍80
␮mol/L (data not shown). Because copper concentrations in the
LDL-containing dialysis bags measured after dilution were ⬇20
␮mol/L, we consider the correction method using EDTA to be
acceptable. The EDTA correction method allows for the endogenous
absorbance of LDL at 480 nm and the very low level of aggregation
of LDL induced by copper during the time scale of the assay (see
Results).
Chemical Modification of ApoB-100
Histidyl residues were modified by incubation with diethylpyrocarbonate, as described by Chen and Frei.22 Briefly, LDL (100 ␮g
protein/mL) was incubated with diethylpyrocarbonate (1 mmol/L) in
PBS for 10 minutes at 37°C. The progress of the reaction and the
amount of modification were monitored spectrophotometrically at
240 nm. After histidyl modification, samples were adjusted to a
density of 1.063 g/mL with a high-density KBr solution and
concentrated to ⬇1 mg protein/mL by ultracentrifugation, as in the
final stages of the LDL isolation method. Lysyl residues were
modified by acetylation with the use of sodium acetate and acetic
anhydride according to the method of Basu et al54 but with additional
acetic anhydride (a total of 10 ␮L/mg LDL protein). Acetylation was
confirmed by demonstrating an increased relative electrophoretic
mobility (REM) with the use of agarose gels, as described below.
Electrophoresis of LDL
Native LDL, LDL carried through the equilibrium dialysis procedure
in the presence of CuSO4 and BHT, oxidized LDL samples, and
chemically modified LDL samples (2 ␮g LDL protein) were allowed
to diffuse into an agarose gel (Paragon Lipo gels, Beckman Instruments) for 5 minutes before the gels were run and stained according
to the manufacturer’s instructions. Destaining was carried out in
ethanol:water (45:55 [vol/vol]) for 1 minute or until background
staining was no longer visible. After the gels were rinsed in water
and dried, the migration of the LDL samples was measured and
divided by the distance migrated by native LDL (REM).
Figure 1. Effect of buffers on copper binding to LDL. Copper
binding to LDL (1 mg LDL protein/mL) was measured at pH 7.4,
in the presence of 10 ␮mol/L CuSO4, 20 ␮mol/L BHT, and either
unbuffered 150 mmol/L NaCl of pH 7.4 (approximately) or
150 mmol/L NaCl with 10 mmol/L phosphate, MES, MOPS,
HEPES, or Tris, all at 4°C and pH 7.4. The number of copper
ions bound per LDL particle (A) and the total concentrations of
copper present in LDL-containing dialysis bags (B, stippled
bars), in dialysis bags without LDL present (B, hatched bars),
and in the bulk dialysis solution (B, open bars) are shown. The
concentrations of copper present in LDL-containing dialysis
bags have been adjusted for 1 mg LDL protein/mL because the
concentration of LDL deviated slightly from that initially present
as a consequence of slight changes in volume in the dialysis
bags. All values shown are the mean⫾SEM of 3 independent
experiments. *Significant (P⬍0.05) inhibition of copper binding
compared with NaCl.
Statistical Analysis
Data are presented as mean⫾SEM of at least 3 experiments, unless
otherwise stated. Results of copper-binding experiments were analyzed by the unpaired 2-tailed t test. Results were considered to be
significantly different at a value of Pⱕ0.05.
Results
Effect of Buffer on Copper Binding to LDL
All buffers tested, except Tris, showed a substantial decrease
in the amount of copper present in solution over 24 hours at
25°C or 37°C, but loss of copper was minimized at 4°C (data
not shown). In MES (pKa 6.3 at 4°C) or MOPS (pKa 7.4 at
4°C) buffers, the copper in solution remained constant over
24 hours at 4°C. The only buffer in which copper concentrations remained constant at all temperatures for 24 hours was
Tris (data not shown). This is probably due to the Tris-copper
complexes formed, although this makes Tris unsuitable for
copper-binding studies. Figure 1 demonstrates the effect of
various buffers on copper binding to LDL. The amount of
copper bound per LDL particle in MES buffer was not
significantly different from the amount bound in MOPS
buffer (Figure 1A). Use of HEPES buffer resulted in a 33%
reduction in copper binding to LDL (P⬍0.001). Phosphate
reduced copper binding by 70% (P⬍0.001) and Tris reduced
binding by 78% (P⬍0.001). Copper binding to LDL in
unbuffered NaCl solution (initially at pH 7.4) was slightly but
Roland et al
Copper Binding to LDL
597
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Figure 2. Aggregation of LDL incubated with CuSO4. LDL (1 mg
protein/mL) was subjected to equilibrium dialysis in Medicell
tubing at 4°C in the presence of MOPS buffer containing 10
␮mol/L CuSO4 and 20 ␮mol/L BHT. Samples of LDL were taken
over a week-long period, and absorbances were measured
(without addition of ascorbic acid or BC) at 480 nm with (open
circle) or without (solid circle) the addition of 1 mmol/L EDTA.
Values were corrected for the measured concentration of LDL
and are shown as the mean⫾SEM of 3 independent experiments. Where the error bars are not shown, they are smaller
than the symbol.
significantly less than that in MOPS buffer (14% reduction,
P⬍0.01). MOPS did not inhibit copper binding to LDL
compared with unbuffered NaCl, suggesting that MOPS does
not bind copper significantly. The slight decrease in copper
binding observed in the absence of a buffer may have been
due to the small drop in pH that was observed, inasmuch as
copper binding to LDL is reduced at acidic pH (authors’
unpublished data, 1998). Total concentrations of copper
within LDL-containing dialysis bags (corrected for 1 mg
protein/mL, because the concentration of LDL can be altered
slightly as a result of volume changes within the bag during
dialysis), copper present in the bulk solution, and copper
present in control dialysis bags without LDL are shown in
Figure 1B. Only when phosphate buffer was used were there
significant decreases in bulk copper concentrations and in the
copper concentrations in LDL-free dialysis bags (P⬍0.01 for
both), which were possibly due to precipitation.
Measurement of Copper-Binding Sites on LDL in
MOPS Buffer
LDL (1 mg protein/mL) was subjected to equilibrium dialysis
with 10 ␮mol/L CuSO4 and 20 ␮mol/L BHT in MOPS buffer
for up to 7 days. Figure 2 shows the attenuation (absorbance
plus light scattering) at 480 nm that was due to LDL, in the
absence of BC and ascorbate. The attenuation increased
greatly after 2 days as a result of light scattering, which
occurred as a consequence of LDL aggregation. Although the
increase was small over the time scale of the copper-binding
assay, during which LDL is exposed to copper for 10 or 24
hours, it can still lead to significant error in the determination
of bound copper. The aggregation became apparent to the
naked eye at late time points, as the LDL became turbid, but
this turbidity was rapidly reversed (as was the light scattering) on the addition of BC and ascorbate (data not shown).
Because the absorbance (A480) values in the presence of BC
and ascorbate have to be corrected by deducting the LDL
absorbance in the absence of BC and ascorbate (LDL has an
absorbance at 480 nm), this light scattering in the absence of
BC and ascorbate could lead to an underestimate of the bound
copper. The addition of 1 mmol/L EDTA to samples without
Figure 3. The effect of CuSO4 concentration, equilibration time,
and LDL concentration on copper binding to LDL. LDL (0.5,
0.75, or 1 mg protein/mL) was dialyzed for up to 24 hours in
Spectra/Por 2.1 dialysis tubing against MOPS buffer containing
0 to 20 ␮mol/L CuSO4 and 20 ␮mol/L BHT. A, Effect of CuSO4
concentration on copper binding to LDL (1 mg protein/mL) over
24 hours. B, Time taken for CuSO4 (10 ␮mol/L) binding to LDL
(1 mg protein/mL) to reach equilibrium. C, Effect of LDL concentration on saturation curves. Data in panels A and B are
mean⫾SEM of 3 independent experiments. Because of the
large amounts of LDL required, data in panel C are means of 2
independent experiments.
BC and ascorbic acid returned the attenuation of light to its
initial level (Figure 2).
The optimum concentration of CuSO4 for use in the copperbinding assay was 10 ␮mol/L, which saturated the copperbinding sites on LDL (Figure 3A). At initial levels of copper of
ⱖ12.5 ␮mol/L, the copper present in the bulk solution decreased
significantly in concentration before the peak copper binding to
LDL was reached, with this effect being most pronounced when
the highest starting concentrations of copper were used (data not
shown). This loss of copper was due to precipitation and was
apparent to the naked eye where starting copper concentrations
between 20 and 100 ␮mol/L were tested.
Figure 3B shows the time taken for copper binding to LDL
to reach equilibrium with use of the Spectra/Por 2.1 dialysis
membrane and 10 ␮mol/L CuSO4. In time-course experiments, a maximum binding of 38 to 42 copper ions per LDL
particle was consistently reached by 10 hours of dialysis. The
same level of maximum binding was obtained after 24 hours
of dialysis across the Medicell membrane. The diffusion of
copper across the membrane to replace that bound by LDL is
presumably the rate-limiting step. The same degree of copper
binding per LDL particle was observed at all tested concen-
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Arterioscler Thromb Vasc Biol.
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Figure 4. Effect of oxidation on copper binding to LDL. Oxidized
LDL samples (1 mg protein/mL) were prepared as described in
the text and were dialyzed in Spectra/Por 2.1 dialysis tubing at
4°C in MOPS buffer with 10 ␮mol/L CuSO4 and 20 ␮mol/L BHT
for 10 hours at pH 7.4. Samples were then analyzed for protein
and copper. The means of 3 independent experiments are
shown as copper ions bound per LDL particle (⫾SEM). *Significant (P⬍0.05) increase in copper binding compared with
control.
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trations of LDL (Figure 3C). The number of copper binding
sites on LDL as the mean of 25 independent experiments (ie,
the mean of 25 equilibrium dialysis copper-binding experiments that we have carried out in the absence of flavonoids or
other inhibitors of copper binding) was 38.6⫾0.7. To ensure
that BHT did not influence copper binding to LDL (other than
by preventing oxidation), experiments were performed that
compared BHT with butylated hydroxyanisole (data not
shown). In the absence of either antioxidant, mean copper
binding to LDL was 50.9⫾4.6 (this high figure was due to the
considerable amount of oxidation that occurred; see below).
The levels of copper binding to LDL observed in the presence
of BHT and butylated hydroxyanisole were not significantly
different (34.3⫾2.1 and 37.2⫾2.7 copper ions per LDL
particle, respectively).
Copper Binding to LDL Isolated From
Different Individuals
LDL was isolated from 12 different volunteers. Copper
binding was measured for each sample in triplicate in a series
of 3 experiments, each with LDL from 4 of the 12 donors.
Individual values for copper binding per LDL particle were as
follows (mean of triplicate): 40.7, 39.8, 39.1, and 37.0; 38.0,
33.6, 35.0, and 35.4; and 38.2, 37.4, 36.5, and 39.2. The mean
of all samples was 37.5, with an SD of 2.1 and an SEM of 0.7.
No significant differences were observed between means of
data for each of the 3 groups of 4 samples each. The
intra-assay coefficient of variation was 4%, and the interassay
value was 5%. These results indicate that the differences
observed between our copper-binding data and the data of
others are not simply due to differences in the LDL samples
used.
Effect of LDL Oxidation on Copper Binding
Although binding assays were carried out at 4°C in the
presence of the antioxidant BHT, it was necessary to consider
the potential effects of LDL oxidation on its copper-binding
properties because of the requirement for relatively long-term
exposure of LDL to copper ions. LDL that had not been
oxidized, mildly oxidized LDL, moderately oxidized LDL
(which contains the peak lipid hydroperoxide levels49), and
highly oxidized LDL (as defined in Methods) differed in their
Figure 5. Inhibition of copper binding to LDL by flavonoids. LDL
(1 mg protein/mL) was dialyzed in MOPS buffer containing 10
␮mol/L CuSO4 and 20 ␮mol/L BHT with various flavonoids (10
␮mol/L). The flavonoids examined are illustrated (A). Flavonoids
were added from stock solutions in ethanol to give a final ethanol concentration of 0.25% (vol/vol). This concentration of ethanol did not affect the copper binding assay. The samples were
then dialyzed in Spectra/Por 2.1 dialysis tubing against MOPS
buffer containing 10 ␮mol/L CuSO4, 20 ␮mol/L BHT, and either
10 ␮mol/L flavonoid or 0.25% (vol/vol) ethanol (control) for 10
hours at 4°C. Histidine (20 ␮mol/L) was included as a positive
control. Copper and protein concentrations were determined.
Results shown are mean⫾SEM of 3 independent experiments
(B). To determine the effect of preincubation with flavonoids on
the copper-binding character of LDL (C), samples were predialyzed in the absence of copper but in the presence or absence
of either quercetin or myricetin (10 ␮mol/L). Predialyzed samples
were then analyzed for copper-binding activity as described
above in the presence or absence of quercetin or catechin (10
␮mol/L). *Significant (P⬍0.05) difference in copper binding compared with control.
copper-binding properties (Figure 4). After 10 hours of
dialysis in Spectra/Por 2.1 tubing, moderately oxidized and
highly oxidized LDL bound significantly more copper than
did native LDL (P⬍0.001), but the binding of copper to LDL
was not increased by mild oxidation. Moderately oxidized
and highly oxidized LDLs were not saturated within the
10-hour time period (data not shown); therefore, the binding
to these oxidized LDLs shown in Figure 4 is an underestimate
of the true level of binding. Data for copper binding to
oxidized LDL at saturation are not presented because at the
long periods of time necessary, the concentration of free
copper declined in the bulk solution, probably as a conse-
Roland et al
quence of precipitation. The extent of oxidation of the
oxidized LDLs and LDL after dialysis in the presence of
copper and BHT was assessed by agarose gel electrophoresis.
REM values were as follows: 1.0 for native LDL and
1.4⫾0.2, 1.9⫾0.1, and 3.1⫾0.2 for mildly, moderately, and
highly oxidized LDL, respectively. The electrophoretic mobility of LDL dialyzed with the copper present consistently
fell between that of native LDL and mildly oxidized LDL
(1.3⫾0.2), implying that LDL is insufficiently oxidized
during the equilibrium dialysis assay to alter its copperbinding properties compared with those of native LDL (see
Figure 4). Lipid hydroperoxides as measured by a triiodide
method55 were not increased during the assay (results not
shown). Thiobarbituric acid–reactive substances were not
suitable indicators of oxidation because they are dialyzable.56
Chemical Modifications of Histidyl and Lysyl
Residues in ApoB-100
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It has been reported that copper binds in part to histidyl
residues in apoB-100, on the basis of results involving their
modification by diethylpyrocarbonate.22,23 Therefore, we
have repeated these experiments, but using our method for
measuring copper binding. Diethylpyrocarbonate was estimated spectrophotometrically to have modified 67⫾4% of
the histidyl residues in apoB-100 and significantly (P⬍0.001)
reduced the number of copper ions bound per LDL particle
from 37.9⫾1.6 to 16.5⫾0.9, a decrease of 56%.
To show that the association of copper ions with LDL, as
measured by our method, was not simply a nonspecific effect of
the net negative charge on LDL electrostatically attracting
positively charged copper ions, we measured copper binding to
acetylated LDL, which has a high net negative charge that is due
to the modification of its lysyl residues.54 Acetylation did not
increase the association of copper with LDL (which would be
expected if the effect were due to the net charge of LDL) and
actually reduced copper binding to 29.1⫾2.2 per particle, a
significant (P⬍0.001) decrease of 23%. The electrophoretic
mobility relative to native LDL was 3.3⫾0.1 for histidylmodified LDL and 4.2⫾0.2 for lysyl-modified LDL.
Inhibition of Copper Binding by Antioxidants:
Effect of Flavonoids
To examine the inhibition by flavonoids of copper binding to
LDL, flavonoids were added immediately from stock solutions in ethanol before the addition of copper to give a final
flavonoid concentration of 10 ␮mol/L and a final ethanol
concentration of 0.25% (vol/vol). As shown in Figure 5B,
myricetin displayed the most potent inhibition of binding
(82% inhibition, P⬍0.001), followed by quercetin (56%
inhibition, P⬍0.001), and catechin (20% inhibition, P⬍0.05).
Epicatechin, kaempferol, and morin at this concentration did
not significantly affect copper binding to LDL. Chrysin was
also tested but was found to cause a significant loss of free
copper from solution by precipitation. Histidine, a compound
with a high affinity for copper ions,57 was included at a
concentration of 20 ␮mol/L to act as a positive control and
was found to reduce copper binding to LDL by ⬎90%.
We examined whether the inhibition by flavonoids of
copper binding to LDL was due to irreversible modification
of copper-binding sites on LDL. LDL samples were predialyzed for 12 hours in Spectra/Por 2.1 tubing in the absence of
Copper Binding to LDL
599
copper, with or without quercetin or myricetin (10 ␮mol/L).
A second dialysis was then performed in the presence of
copper as usual (Figure 5C). For samples that had been
preincubated with flavonoids, this second dialysis was carried
out in the absence of flavonoids. For samples that had been
preincubated in the absence of flavonoids, quercetin or
myricetin (10 ␮mol/L) was included in the second dialysis.
Control LDL did not encounter flavonoids at either dialysis
stage. LDL that had been preincubated with either quercetin
or myricetin bound significantly more, rather than less,
copper than LDL that had been preincubated in the absence of
flavonoids (P⬍0.05), when quercetin or myricetin was absent
from the second dialysis. This suggests that copper can form
a tertiary complex with LDL-bound flavonoids but can be
easily removed from LDL or LDL-associated flavonoids by
free flavonoids in solution.
Discussion
Our results demonstrate the presence of 38.6⫾0.7 (n⫽25
independent experiments) copper-binding sites per LDL particle. MOPS/NaCl was the most suitable buffer for the
measurement of copper binding to LDL by equilibrium
dialysis. Tris, phosphate, and, to a lesser extent, HEPES
presumably formed complexes with copper ions and thereby
reduced copper binding to LDL. HEPES and Tris have been
reported to have a low affinity for copper, as determined by
displacement of a pH titration curve when copper was present
at a concentration equivalent to that of the buffer.58 In
systems used to measure copper binding to LDL, however,
the buffer is present in great excess over the copper (1000fold excess in our system). Therefore, a buffer normally
considered to have a low affinity for copper can still interfere
significantly with copper-binding assays. We cannot exclude
the possibility that some of the buffers that inhibit copper
binding to LDL may do so by blocking copper-binding sites
on the lipoprotein.
The number of binding sites increased with LDL oxidation,
although the low level of oxidation occurring during the
equilibrium dialysis procedure with copper in the presence of
BHT was not sufficient to affect the copper binding properties of LDL (Figure 4). Increased binding of divalent cations,
such as manganese(II), by LDL on oxidation or malondialdehyde modification has been reported by others59 and has
been suggested to be due to the modification of lysyl residues,
which may prevent their interaction with aspartyl and glutamyl residues in apoB-100 and thus increase the binding of
divalent cations to these acidic residues. However, this does
not explain the increased copper-binding capacity of oxidized
LDL, inasmuch as the acetylation of lysyl residues significantly decreased copper binding to LDL. The decrease in
copper binding associated with acetylation (and the concomitant increase in net negative charge) of LDL also suggests
that the increased copper-binding capacity of oxidized LDL is
not purely charge dependent. It is possible that increased
copper binding by oxidized LDL is a consequence of the
proteolysis of apoB-100 that occurs during oxidation,60 which
may facilitate conformational changes that result in the
exposure of new sites with copper-binding potential. It is also
possible that copper may bind to the carboxylate groups of
fatty acid derivatives generated by lipoprotein-associated
600
Arterioscler Thromb Vasc Biol.
April 2001
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phospholipase A2 during LDL oxidation61,62 or to oxidatively
modified aminoacyl residues in apoB-100.63
We have demonstrated a 56% reduction in copper binding
after the modification of histidyl residues in apoB-100 and
have thereby confirmed the findings of Chen and Frei22 and
Wagner and Heinecke.23 Acetylation of lysyl residues resulted in a 23% reduction in copper binding. This might
conceivably be due to a conformational change in apoB-100.
Copper ion binding to the enol ether double bond of plasmalogens may account for some of the remaining copperbinding sites on LDL.64
As far as we are aware, we report the first reversible
copper-dependent aggregation of LDL. LDL became aggregated during prolonged incubation with copper in the presence of BHT. BC and EDTA were both capable of completely
reversing the aggregation. Although the mechanism of this
aggregation is unclear, it may be related to the reversible
copper-dependent aggregation of amyloid-␤ peptides.65
The number of copper binding sites previously reported on
LDL varies enormously and appears to depend primarily on
the choice of buffer and the method of separating bound from
free copper (Table). Separation by ultrafiltration22,23,27,28 has
led to estimates quite different from ours and may be
confounded by interactions of copper ions with LDL gel
layers. Gel-layer formation results in a barrier to filtration
above the membrane, greatly decreasing the speed of ultrafiltration and making the passage of small molecules into the
filtrate increasingly difficult.66,67
Column chromatography gives very low estimates of
copper binding to LDL.24,26,30 There is likely to be a problem
with this technique, inasmuch as copper ions bind to Sephadex during column chromatography. This can be readily
seen by passing LDL-copper complexes through Sephadex
G-25 PD-10 columns (Pharmacia Biotech) and eluting with
BC and ascorbate after the LDL has emerged and the column
has been eluted with several times its bed volume.
Copper-BC complex formation is visible only at the head of
the column, even if the BC/ascorbate solution is passed
through the column in reverse, ie, from the foot upward.
Precipitation of LDL with the use of methanol and HDL
cholesterol precipitant has also been used to separate free
from lipoprotein-bound copper31 and gives an estimate of
⬇100 copper ions bound per LDL particle. This technique
may have measured copper binding to denatured LDL, and it
has been speculated that copper may have been adsorbed to
the precipitate.32
Kinetic analysis based on the oxidation of LDL by copper
has been used to examine copper binding to LDL,32,68,69 but
this would measure only redox-active binding sites.
Using dialysis to separate free from lipoprotein-bound
copper in the absence of a bulk pool of copper in the dialysis
buffer26,29 may result in the dissociation of some of the copper
from the LDL.
Equilibrium dialysis has been used by other workers,23 but
the procedure was carried out in a HEPES buffer, which we
find decreases the binding of copper to LDL (Figure 1). Also,
the dialysis was performed for only 4 hours, which may have
been insufficient to saturate the copper binding sites on LDL.
For the reasons given above, we believe that the equilibrium dialysis technique described in the present study has
advantages over other methods that measure copper binding
to LDL.
Quercetin and myricetin substantially inhibited copper
binding to LDL, with myricetin being significantly more
effective than quercetin (P⬍0.001). These 2 flavonols possess a carbonyl group at the 4 position and hydroxyl groups
at the 3 and 5 positions. Either of these hydroxyl groups could
act in concert with the carbonyl oxygen to chelate transition
metal ions.70 However, kaempferol and morin were unable to
inhibit copper binding to LDL despite the presence of a
carbonyl group at C-4 and hydroxyl groups at C-3 and C-5,
suggesting that these features alone are insufficient to allow
significant copper binding to a flavonoid. Adjacent hydroxyl
groups on the B ring have also been implicated in transition
metal binding by flavonoids,47,71,72 but these are present in
catechin and epicatechin (both of which lack a C-4 carbonyl
and a C-2 to C-3 double bond) without enabling these
flavan-3-ols to bind copper to any great extent at the
concentration used in the present study. It is likely that the
C-ring flavone structure acts in concert with B-ring vicinal
hydroxyl groups to facilitate copper complex formation by
the effective flavonoids, possibly after hydrogen abstraction
from a B-ring hydroxyl group.70 The superior ability of
myricetin over quercetin to decrease copper binding to LDL
may be due to the presence of the additional hydroxyl group
on the B ring, providing additional stabilization of a 4⬘ anion
through hydrogen bond formation and through resonance
stabilization. Also, the presence of 3 sequential hydroxyl
groups in the B ring increases the probability of copper
chelation by 2 adjacent oxygen atoms.
The antioxidant activity of flavonoids depends on their free
radical–scavenging activity as well as their ability to chelate
copper or iron, inasmuch as kaempferol and morin inhibit
lipoprotein oxidation34,35,47 even though they do not inhibit
copper binding to LDL. Once attacked by free radicals,
oxidized flavonols might continue to act as antioxidants by
chelating pro-oxidant metal ions and holding them in an
inactive form.
The occurrence of transition metal ion binding by flavonoids in vivo remains to be determined, but sequestration
of pro-oxidant metal ions may play a part in the possible
protection against atherosclerosis by dietary flavonoids. Because flavonoids can be extensively metabolized in vivo,73
the ability of flavonoid metabolites to bind transition metals
warrants examination. It has been suggested that by analogy
with other inflammatory sites, atherosclerotic lesions may
have an acidic extracellular pH, and it is known that some
metal-carrying proteins release transition metal ions under
acidic conditions.17 Because pH affects metal binding to
flavonoids,44,71,74 studies could be focused on flavonoids
having structures that are likely to confer the highest metalbinding activity at mildly acidic pH.
Acknowledgments
This work was supported by a research studentship (A.R.) from the
Ministry of Agriculture, Fisheries and Food and by a grant from The
Wellcome Trust (R.A.P. and D.S.L.). The authors wish to thank
Andrew P. Brown for carrying out preliminary aminoacyl modification experiments; the Nuffield Foundation for an undergraduate
research bursary (A.P.B.); Mehregan Movassagh, Justin P. Richards,
and Sohail Mushtaq for their excellent technical assistance; Dr V.
Roland et al
Moens at the University of Reading Health Center for skillful
venipuncture; and the volunteers who gave blood.
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Measurement of Copper-Binding Sites on Low Density Lipoprotein
Alexander Roland, Rebecca A. Patterson and David S. Leake
Arterioscler Thromb Vasc Biol. 2001;21:594-602
doi: 10.1161/01.ATV.21.4.594
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