From the Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santa Cruz, California 95064
Received for publication, December 20, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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Congo red (CR) binding, monitored by
characteristic yellow-green birefringence under crossed polarization
has been used as a diagnostic test for the presence of amyloid in
tissue sections for several decades. This assay is also widely used for
the characterization of in vitro amyloid fibrils. In order
to probe the structural specificity of Congo red binding to amyloid
fibrils we have used an induced circular dichroism (CD) assay. Amyloid
fibrils from insulin and the variable domain of Ig light chain
demonstrate induced CD spectra upon binding to Congo red. Surprisingly,
the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than
those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to
representative proteins from different classes of secondary structure
such as In the 1920s Benhold (1) and Divry (2) established that Congo red
bound to amyloid in tissue sections and demonstrated its characteristic
yellow-green birefringence under crossed polarizers. Since then this
birefringence has been used as a diagnostic for amyloid fibrils. The
birefringence assay is not a simple one, for example, the tissue
sections need to be of a required thickness to show birefringence,
reviewed by Elghetany and Saleem (3) and Westermark et al.
(4). Congo red binding is not specific for amyloid in the tissue
sections, but the assays are performed under extreme conditions with
50-80% ethanol, high salt and alkaline pH conditions to yield binding
to amyloid (5). Despite these extreme conditions, binding to collagen
fibers and cytoskeletal proteins results in false-positive results (4).
Due to the difficulties with the birefringence assay for in
vitro amyloid fibrils, Klunk and co-workers (6, 7) have developed
simpler filtration based assays followed by measuring the concentration of free Congo Red to quantify dye binding. The filtration assays would
not detect CR1 bound to
soluble monomers or oligomers as they are not large enough to be
trapped by 0.2-µm filters. Large particles, such as amyloid fibrils,
are retained on the filters accounting for the loss of free dye
molecules, whereas any native protein molecules bound to CR would pass
through the filter pores. Thus the filtration assay is not affected by
possible binding of CR to native soluble conformations of protein.
Benditt and co-workers (8) analyzed spectral probes such as absorbance
red shift, optical rotary dispersion, and circular dichroism
(CD) to describe the Cotton effect (9) responsible for birefringence.
They used human albumin, poly-L-lysine with different
conformations, and amyloid fibrils as substrates for binding of Congo
red. The random coil conformation of poly-L-lysine did not
show spectral changes, but both helical and Edwards and Woody (10, 11) demonstrated that induced circular dichroism
can be used as a probe for Cibacron blue and Congo red bound to
dehydrogenases such as liver alcohol dehydrogenase, yeast alcohol
dehydrogenase, lactic dehydrogenase, and kinases including
phosphoglycerate kinase and porcine adenylate kinase. Edwards and
Woody (11) believed that Congo red bound to the coenzyme-binding sites
of the enzymes based on the similarities between dye and coenzyme
structures. Congo red has also been shown to bind other native proteins
including cellular prion protein (12), elastin (13), RNA polymerase
(14), and human prostatic phosphatase (15).
Structure T1 of Congo red suggests that
binding to protein could occur through a combination of both
hydrophobic and electrostatic interactions. An additional complication
is that CR has been reported to form linear ribbon-like micelles (16,
17). In an attempt to understand the binding specificity of Congo red
we have used induced circular dichroism as an assay for binding of CR
to native proteins, partially folded protein conformations, and amyloid fibrils, using native proteins from a variety of different secondary structure classes. The results suggest a mechanism of binding of Congo
red to native proteins involving the intercalation of Congo red between
protein molecules leading to oligomerization of the protein.
Fibril Formation--
Fibril were grown in vitro with
a 0.5 mg/ml SMA (a recombinant amyloidogenic variable domain of Ig
light chain made by Stevens and co-workers (18)) and 1 mg/ml bovine
insulin solution at pH 2 in 20 to 50 mM HCl and 100 mM NaCl, that were agitated using a magnetic stirrer in a
37 °C incubator for a day.
CD Measurements--
CD spectra were collected between 650 and
300 nm, with 1-nm step size, 10 s averaging time, in an Aviv 62 DS
spectropolarimeter. Induced CD spectra were obtained using a split
quartz cell with each compartment having a path length of 5 mm and
total path length of 10 mm. The protein and Congo red solutions of
twice the final concentration were added in each compartment to obtain
a control spectrum. Induced circular dichroism was only observed upon
mixing the Congo red and the protein solutions before collecting the spectrum again. The induced CD assay was performed at pH 7.5 using a
1-cm path length rectangular cell with final Congo red concentration of
30 to 40 µM with protein concentration of 0.1 to 0.2 mg/ml. At pH 2.0 the induced CD assay was performed using a 10-cm path length circular CD cell with final Congo red concentrations of 3-4
µM as the solubility of Congo red was much lower at pH 2. Induced CD measurements for fibril samples were obtained with a 10-cm
path length circular cell using very dilute samples (80 to 400 nM), as at higher concentrations the fibril samples
precipitated as red particles.
Attenuated Total Reflectance-Fourier Transform Infrared
Spectroscopy (ATR-FTIR)--
Hydrated (H2O) thin film
spectra were collected using a Nicolet 800 FTIR spectrometer equipped
with a liquid nitrogen-cooled MCT detector and purged with dry air. All
samples were scanned in an out-of-compartment horizontal ATR accessory
(SPECAC) with a high throughput 73 × 10 × 6 mm, 45°
trapezoidal germanium crystal (the internal reflectance element). To
collect spectra protein samples with and without Congo red were applied
(40 µl of 1 mg/ml protein solution and 500 µM Congo
red) and dried while being spread constantly with a spatula. Data
processing was done with GRAMS32 (Galactic Industries) and SAFAIR
software as previously described (19). Water vapor components were
subtracted until the region between 1800 and 1700 wavenumbers was featureless.
Analysis of Binding Constants and Number of Binding
Sites--
Equation 1 was used to fit the data obtained at 40 µM Congo red and varying the protein concentration from 0 to 20 µM and measuring the CD spectra of protein bound
dye molecules.
Cross-linking and SDS-PAGE Analysis--
10 µM
DTSSP (3,3'-dithiobis(sulfosuccinimidylpropionate)), a bifunctional,
cleavable cross-linking reagent, was added to the protein solution in
the absence and presence of Congo red and incubated for 1 h. DTSSP
(Pierce) is a sulfonated derivative of DSP
(dithiobis(succinimidylpropionate)) that is water soluble, unlike DSP,
and, like DSP, can be cleaved under reducing conditions (the presence
of dithiothreitol or Small Angle X-ray Scattering Experiments--
Small angle x-ray
scattering (SAXS) measurements were done using Beam Line 4-2 at the
Stanford Synchrotron Radiation Laboratory. X-ray energy was
selected at 8980 eV (Cu edge) by a pair of Mo/B4C multilayer monochromator crystals. Scattering patterns were
recorded by a linear position-sensitive proportional counter, which was filled with an 80% Xe, 20% CO2 gas mixture.
Scattering patterns were normalized by incident x-ray fluctuations,
which were measured with a short length ion chamber before the sample.
The sample-to-detector distance was calibrated to be 230 cm, using a
cholesterol myristate sample. The measurements were performed in a
1.3-mm path length observation static cell with 25-µm mica windows.
To avoid radiation damage of the protein samples during the SAXS
measurements, the scavenger
N-tert-butyl-
The radius of gyration (Rg) was calculated
according to the Guinier approximation (41),
Estimation of the Shape of Particles using SAXS Data--
The
relationship between RS (Stokes radius) and
Rg (radius of gyration) is quite sensitive to the
shape and compactness of protein, as
RS/Rg =
P1/3[5/(P2 + 2)]1/2, where P = a/b, and
a and b are the semiaxis of the revolution of the
ellipsoid and the equatorial radius of the ellipsoid, respectively (20-22). In order to estimate the RS value for the
oligomeric form of Amyloid Fibrils and Native Conformations Show Different Induced
Congo Red CD Bands--
Amyloid fibrils made in vitro from
purified proteins such as bovine insulin and Ig light chain (SMA), were
tested for binding to Congo red using the apple-green birefringence
under crossed polarization (data not shown) and induced CD signal (Fig.
1A). Low concentrations of
fibril solutions were used for induced CD measurements, as higher
concentrations yielded red precipitates. Both fibril samples showed
major induced CD bands with positive maxima in the vicinity of 570 nm
and negative maxima in the vicinity of 500-525 nm. The spectrum for
the induced CD of insulin fibrils was red-shifted relative to that for
the light chain fibrils. The shape of the induced CD bands for Congo
red bound to fibrils is different from that described by Benditt
et al. (8) for tissue-extracted amyloid. The differences are
most likely due to "contaminants" such as glycosaminoglycans,
serum amyloid protein etc. present in the ex vivo extracts.
Native insulin and native SMA show induced Congo red CD bands that
differ in shape and intensities from the CD bands induced upon binding
to the fibrils (Fig. 1B). Instead of a positive and a
negative peak observed for the corresponding amyloid fibrils the native
proteins show a broad positive band between 500 and 600 nm, that
possibly has several components. The peculiar CD band shape with a
maxima and a minima is related to the special nature of birefringence
(26) observed for amyloid fibrils upon binding to Congo red. The
positive peaks induced by the native proteins were 10-fold smaller in
intensity compared with their corresponding amyloid fibrils. A probable
explanation is that amyloid fibrils have more binding sites for Congo
red than the native protein. The intensity of the induced Congo red CD
bands upon binding to native SMA was much smaller than that for native
insulin. It is possible that the hexameric insulin (under native
conditions) has more Congo red binding sites, leading to a larger
induced CD signal, compared with native SMA.
Congo Red Binds to Native Proteins from Different Secondary
Structure Classes--
Native proteins such as lysozyme (
Given that unfolded protein did not induce Congo red CD bands, we
conclude that significant secondary structure, and probably a collapsed
conformation is required to induce a specific orientation of Congo red
molecules responsible for CD bands. Indeed Turnell and Finch (27)
observed a Congo red molecule intercalated between the antiparallel
In view of the fact that the induced CD spectra demonstrate that
proteins from all classes of secondary structures bind Congo red, Congo
red binding is clearly not restricted to the crossed- No Secondary Structure Changes Observed in Proteins upon Binding of
Congo Red--
Since it has been suggested that Congo red bound
specifically to crossed Native and Partially Folded Conformations Induce Different Congo
Red CD Bands--
1,8-Anilinonaphthalene sulfonate (33) and its dimer
bis-1,8-anilinonaphthalene sulfonate (34) are commonly used as probes of hydrophobic regions in native and partially folded proteins. Due to
the similarity of the structures of Congo red and
bis-1,8-anilinonaphthalene sulfonate we decided to also test
differential binding of Congo red to native and partially folded
conformations. Apomyoglobin exists in its native conformation at pH 7, the acid unfolded state at pH 2 in the absence of salts and as a
partially folded intermediate at pH 4 (35, 36), and at pH 2 in the
presence of salt (37). No induced Congo red CD bands were observed for
the acid unfolded form, but the native (pH 7) and partially folded
conformations at pH 4 and 2 with 500 mM KCl showed
different induced spectra (Fig. 5).
Binding of the dye to the native conformation of apomyoglobin is not
surprising, since the protein is known to bind a variety of hydrophobic
molecules in the vacant heme-binding site. Increased binding of Congo
red has been observed for the molten globule intermediate compared with
the native conformation for human prostatic phosphatase (7 to 8 molecules of Congo red bind to the intermediate conformation as opposed
to 1.6 dye molecules to the native protein) (15). Consequently it
appears that CR-binding sites are present in partially folded
intermediates. This is not surprising since such intermediates are
known to have exposed hydrophobic patches and bind hydrophobic
molecules. Thus it is likely that different binding sites for CR may
exist in native and partially folded intermediate states.
Probing the Mechanism of Congo Red Binding--
The number of
molecules of Congo red bound per molecule of native Oligomerization of Proteins upon Congo Red Binding--
To test if
Congo red binding involved oligomerization of protein molecules we
added DTSSP, a cleavable cross-linker, to the protein solution in the
absence and presence of Congo red. Analysis by cross-linking and
nonreducing SDS-PAGE further confirmed the binding of Congo red to many
proteins (Fig. 7A). The
control experiment in which the protein was cross-linked in the absence
of Congo red revealed mostly monomeric species. The Congo red-bound
protein bands showed up as red bands in the nonreducing gels until the gel was stained with Coomassie Brilliant Blue dye. This was due to the
acidic conditions since Congo red is a pH indicator and turns blue at
low pH. Upon drying and removal of acetic acid, the Congo red bands
turned red again. The slower migration of the cross-linked Congo
red-bound protein suggests that the proteins oligomerized during or
after binding Congo red. Cleavage of the cross-linker under reducing
conditions revealed only monomeric protein bands and free Congo red
(Fig. 7B). Free Congo red runs as a red band approximately
the size of a 50-kDa protein. Congo red has been reported to
self-associate and form ribbon-like micelles (39, 40). It is possible
that in the presence of sodium dodecyl sulfate, Congo red is a
self-associated oligomer that runs as a high molecular weight
species.
The results show that CR causes association of a variety of native
proteins with different classes of secondary structure. The most
reasonable explanation is that CR bridges two molecules by
intercalating between hydrophobic surface patches with appropriate electrostatic regions for the sulfonate groups.
Size and Shape of Congo Red Bound Oligomers--
Small angle
scattering of x-rays by protein molecules can provide information about
their size, shape, and globularity (41). One of the most commonly used
applications of SAXS is measurement of the size of a scattering
molecule using Guinier analysis, which is based on the Gaussian shape
of the scattering curve near zero angles. The Guinier plot for a
homogeneous system is generally linear at small angles, allowing
estimation of the radius of gyration of the particle,
Rg (41). Fig.
8A represents the
Guinier plots for bovine
The Kratky plot, I(S) × S2 versus S, is a useful
expression to describe the structural characteristics of a polymer
(41). It has been shown that the shape of the Kratky plot is sensitive to protein conformation (42-45). In particular, for native globular proteins the Kratky plot has a characteristic position for the maximum
that depends on the dimensions of the scattering particle, and shifts
to smaller angles with an increase in Rg (41). Fig.
8B represents Kratky plots for
The hydrodynamic data allow us to estimate the shape of the
Birefringence Assay in ex Vivo Tissue Sections--
Congo red
staining is a standard method used to examine ex vivo tissue
sections for amyloid fibril deposits. The ex vivo tissue sections are tested for the presence of amyloid by first denaturing native proteins in the tissue sections followed by staining the fibrils
with Congo red. These sections are then tested for birefringence under
crossed polarization using light microscopy (5). The results
described here show that Congo red binds to many native proteins and
lacks secondary structure specificity. This would explain the
false-positive results obtained in tissue sections with cytoskeletal
proteins that are stable under the conditions used for Congo red
staining in tissue sections. This further confirms that specific
crossed Model of Congo Red Binding--
Although Congo red is well
established as an inhibitor of fibril formation for several proteins
(e.g. A
It has been reported that Congo red forms long rod-like "micelles"
due to parallel stacking of the aromatics groups (17), and that these
supramolecular forms of the dye specifically interact with (citrate synthase),
+
(lysozyme),
(concavalin A), and parallel
-helical proteins (pectate lyase). Partially folded
intermediates of apomyoglobin induced different Congo red CD bands than
the corresponding native conformation, however, no induced CD bands
were observed with unfolded protein. Congo red was also found to induce
oligomerization of native proteins, as demonstrated by covalent
cross-linking and small angle x-ray scattering. Our data suggest that
Congo red is sandwiched between two protein molecules causing protein
oligomerization. The fact that Congo red binds to native, partially
folded conformations and amyloid fibrils of several proteins shows that
it must be used with caution as a diagnostic test for the presence of
amyloid fibrils in vitro.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
conformations induced
Congo red CD bands as well as optical rotary dispersion spectra similar
to those observed with the amyloid fibril samples.
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Structure 1.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
Where C is the change in circular dichroism signal at
a particular wavelength measured upon addition of protein
([L]) in varying concentration, Cm is
the maximal CD change obtained by varying the protein concentration at
a constant Congo red concentration, n is the number of
binding sites for Congo red on the protein, and Kd
is the dissociation constant for binding of Congo red to protein.
(Eq. 1)
-mercaptoethanol). The cross-linked protein was
then precipitated using 10% acetic acid, and spun in a centrifuge. The
pellet was resuspended in SDS loading buffer and separated on either a
nonreducing or a reducing 8-25% SDS-PAGE gel.
-(4-pyridyl)-nitrone-N'-oxide
was added to a final concentration of 10 mM. Background
measurements were performed before and after each protein measurement
and then averaged for background subtraction. All SAXS measurements
were performed at 23 ± 1 °C.
where Q is the scattering vector given by
Q = (4
(Eq. 2)
sin
)/
(2
is
the scattering angle, and
is the wavelength of x-ray). I(0), the forward scattering amplitude, is proportional to
n·
c2·V2,
where n is the number of scatterers (protein molecules) in
solution;
c is the electron
density difference between the scatter and the solvent; and
V is the volume of the scatter. This means that the value of
forward-scattered intensity, I(0), is proportional to the
square of the molecular weight of the molecule (41). Thus,
I(0) for a pure N-mer sample will
therefore be N-fold that for a sample with the same
number of monomers since each N-mer will scatter
N2 times as strongly as monomer, but in
this case the number of scattering particles (N-mers)
will be N times less than that in the pure monomer sample.
-lactoglobulin the following approach was used.
SAXS data show that in the presence of 1 mM Congo red
-lactoglobulin forms 28-mers, which corresponds to particles with
the molecular mass of 515,200 Da (= 28 × 18, 400 Da). The value
of RS of a globular protein with the molecular mass
of 515,200 Da is 71.4 Å. This was calculated from an empirical
equation log = 0.369·log(M)
0.254 (23) based on
the intrinsic viscosity data (24). Here R
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Induced circular dichroism spectra of Congo
red on binding to native or fibrillar proteins. A,
induced CD spectra on binding of 40 µM Congo red to 80 nM insulin fibrils ( ) and 400 nM Ig light
chain fibrils (- - - -), and Congo red alone (···), in a 10-cm
path length circular cell. Amyloid fibrils were made in
vitro by stirring solutions of insulin and Ig light chain: the
presence of fibrils was confirmed with EM or atomic force microscopy.
B, induced CD spectra of 40 µM Congo red with
10 µM native insulin (
) or native Ig light chain
(- - - -). Lower concentrations of fibrils were used to prevent
formation of red precipitates.
+
),
concavalin A (
), and citrate synthase (all
) (Fig.
2) showed induced Congo red CD signals,
thus revealing binding of Congo red. A number of other native proteins
from different structural classes, interleukin-2 (all
), malate
dehydrogenase (
/
),
-lactoglobulin, and apomyoglobin (
) also
showed induced Congo red CD signals (data not shown). Other than the
observation that the major induced CD bands were in the vicinity of 525 nm, and quite broad, no consistent patterns were apparent. Circular
dichroism bands in the visible region were not observed for protein
alone, or Congo red alone. This was demonstrated by using split cells
where one side of the cell contains the protein solution and the other
side of the cell contains Congo red solution, no CD bands were observed
until the protein and Congo red solutions were mixed together.
Interestingly, no Congo red CD bands were induced in the presence of
unfolded protein (e.g. acid unfolded apomyoglobin; Fig. 5),
amorphous aggregates of P22 tail spike protein, or Ig light chain
variable domain, or inclusion bodies. These results are supported by a
report that there are no Congo red CD or optical rotary dispersion
signals with the random coil conformation of poly-L-lysine
(8). Whereas the induced CD bands indicate a specific orientation of
Congo red, which is assumed to be due to binding of the dye to the
protein, the absence of an induced CD band does not necessarily mean
the absence of dye binding, but rather the lack of specific orientation responsible for the induced CD bands.
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Fig. 2.
Induced CD spectra of Congo red (40 µM) with citrate synthase (an
all- protein,
), lysozyme (an
/
protein,
),
and concavalin A (an all-
protein,
- - - -) under native conditions, 2 µM protein, 20 mM phosphate, pH 7.5, and 150 mM NaCl, after 30 min incubation. The
dotted line represents the spectra of unmixed solutions of
80 µM Congo red and 4 µM protein in a split
cuvette.
-strands of two insulin molecules, using x-ray diffraction of a
crystalline complex. However, our results suggest that Congo red
binding is not limited to
-sheet proteins; rather, all classes of
proteins,
-helical,
, and
/
, also bind Congo red resulting
in the observed induced CD bands. Congo red binding to dehydrogenases
of the
/
class has been reported previously, and is thought to
reflect specific interactions with coenzyme-binding sites, due to
structural similarities between CR and the coenzyme (10).
-Helical
proteins also induced Congo red CD bands (Fig. 3). Interestingly, the right-handed
-helical proteins including pectate lyase (28), and p22 tailspike
protein (29) induced different Congo red CD bands, with positive
ellipticity, compared with the left-handed
-helical protein LpxA
(30), which induced two negative Congo red CD bands. This suggests that
the positive or negative CD bands may reflect the underlying chirality
of the CR-binding site.
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Fig. 3.
Induced CD spectra for two right-handed
-helical proteins, pectate lyase (
··
··)
and P22 tailspike protein (- - - -), and a left-handed
-helical protein LpxA (
). Aggregated P22
tailspike protein (···) does not bind Congo red, and the
resulting spectrum corresponds to that of Congo red alone in the
absence of protein.
structures
present in amyloid fibrils. Benditt and co-workers (8) have also shown
binding of Congo red to both
and
conformations of
poly-L-lysine. It is possible that the shape of the induced Congo red CD bands has specific clues as to which secondary structures in the proteins Congo red dye is bound, but more work is needed to
understand these distinctions. The lack of correlation between the
shape of the induced CD band and the protein secondary structure suggests that binding sites for CR in individual proteins are more
related to their specific environment rather than to a particular type
of secondary structure.
structures present in amyloid fibrils (31),
or to
-sheets in native proteins (32), and our results indicated that all
proteins also bind Congo red, we sought to confirm that
Congo red binding does not induce changes in secondary structure, e.g. from
to
. To test this we collected infrared
spectra of interleukin-2 (IL-2) (a four-helix bundle protein) in the
absence and presence of Congo red (Fig.
4A). The spectrum of Congo red alone was featureless in the amide I and II regions where proteins show
specific conformational-sensitive bands, and no secondary structure
changes were observed in the IL-2 FTIR spectrum upon binding to Congo
red (Fig. 4A). Lysozyme, an
/
protein, forms a red
precipitate upon binding to Congo red. To test if this precipitate involves formation of new
-structure with low wavenumber amide I
peaks, as observed for many protein
aggregates,2 we examined it
with ATR-FTIR. The FTIR spectra of soluble free lysozyme and
precipitated Congo red-bound lysozyme are compared in Fig.
4B. No significant increase in
-structure was observed in
the precipitated Congo red-bound lysozyme compared with native lysozyme. The minor differences observed between 1610 and 1580 cm
1 are probably due to interaction of Congo red with
specific side chains, as these bands have significant contributions
from side chains and are not indicative of protein secondary structure
changes. Thus it is clear that binding of CR does not result in
induction of
-structure.
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Fig. 4.
FTIR spectra reveal that Congo red does not
induce -structure on binding to protein.
A, ATR-FTIR spectra of IL-2 (an all-
-protein) showing the
amide I and amide II regions with (- - - -) and without Congo red
(
). B, lysozyme forms a red precipitate upon incubation
with Congo red (1 mg/ml lysozyme with 0.5 M Congo red), and
the ATR-FTIR spectrum for this precipitate (- - - -) is compared
with the spectrum for soluble native lysozyme (
).
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Fig. 5.
Native apomyoglobin shows an induced CD band
upon binding to Congo red ( ). Acidic unfolded apomyoglobin at pH
2 with no salt (···) shows no CD bands. Acidic partially folded
intermediates stabilized at pH 2 in the presence of salt
(
··
··) and at pH 4 (- - - -) show induced CD bands that
are different from the ones obtained from native apomyoglobin.
-lactoglobulin
was estimated from an analysis of the data obtained by varying the
concentration of protein from 0 to 20 µM while keeping
the concentration of Congo red constant at 40 µM (Fig.
6A). The ellipticity at 530 and 450 nm were plotted against
-lactoglobulin concentration (Fig.
6B) and the data were fitted to Equation 1. The analysis
showed that 1.52 ± 0.05 molecules of Congo red bound per molecule
of
-lactoglobulin, similar to the value obtained by Kuciel and
Mazurkiewicz (15) for human prostatic phosphatase. Since we could not
measure the concentration of unbound protein for technical reasons, a
more accurate analysis involving Scatchard plots was not possible. A
likely mechanism would involve three protein molecules with two Congo
red molecules intercalated between them. The interaction of Congo red
with protein molecules may involve a complex of multiple protein
molecules with intercalated Congo red molecules. Intercalation as a
mechanism of binding of Congo red molecules between peptide chains has
also been suggested by Stopa and co-workers (38).
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Fig. 6.
Titration of Congo red and
-lactoglobulin. A, induced circular
dichroism spectra of 40 µM Congo red increasing
concentrations of
-lactoglobulin: 0.5 µM (
), 2.0 µM (···), 5.0 µM (- - - -), 10 µM (
), and 20 µM (
·
·
).
B, the ellipticity at 530 nm (
) and 450 nm (µ) plotted
against
lactoglobulin concentration and fitted to Equation 2,
indicating that 1.52 ± 0.05 molecules of Congo red bound per
molecule of
-lactoglobulin.
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Fig. 7.
Congo red induces oligomerization of native
proteins. A, 8-25% nonreducing SDS-PAGE after
cross-linking with DTSSP (a cleavable amine-reactive cross-linker), in
the absence ( ) and presence (+) of Congo red for pectate lyase,
carbonic anhydrase (CA),
-lactoglobulin, IL-2, and
lysozyme under nonreducing conditions. B, the same samples
but after the cross-linker was cleaved by reducing the disulfide bond.
Free Congo red (red bands) and monomeric protein bands
(stained with Coomassie Brilliant Blue) appear.
-lactoglobulin (250 µM)
measured in the absence and presence of different Congo red
concentrations (50 µM and 1 mM). All plots
are linear functions, reflecting the fact that all three systems are
essentially homogeneous, i.e. monodisperse. Dimensions of
the protein are unaffected by the addition of small amounts of Congo
red (Rg = 18.7 ± 0.3 and 18.7 ± 0.3 Å in the presence and absence of 50 µM Congo red,
respectively). However, in the presence of 4-fold excess of the dye
over the protein, the Rg value increases about
3-fold (Rg = 56.5 ± 0.9 Å), reflecting the
Congo red-induced association of
-lactoglobulin. Additional
information on the degree of protein association could be extracted
from the analysis of the forward scattered intensity values. The
addition of 1 mM Congo red to 250 µM
-lactoglobulin results in a 28-fold increase in the I(0) value, indicating Congo red-bound
-lactoglobulin forms large oligomers (i.e. ~28-mers).
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Fig. 8.
Small-angle x-ray scattering analysis of the
size of complexes of Congo red with
-lactoglobulin. A, Guinier plots
for 250 µM
-lactoglobulin alone (
), and in the
presence of 50 µM (
) and 1 mM Congo red
(
). The increased slope at high CR concentration indicates a
significantly larger Rg. B, Kratky plots
for
-lactoglobulin measured in the absence (
) and presence of two
different concentrations of Congo red, 50 µM (···)
and 1 mM (
). Both plots demonstrate that the
presence of Congo red (4-fold higher concentration than the protein)
induces oligomerization of the
-lactoglobulin.
-lactoglobulin in the
absence and presence of two different concentrations of Congo red.
Curves for
-lactoglobulin in the absence or presence of 50 µM Congo red are superimposable and show the
characteristic symmetric maximum of a native globular protein. However,
the scattering profile for the protein in the presence of 1 mM Congo red (4-fold excess of protein) shows two very
distinct maxima. The position of one of these maxima is comparable to
that of the curves in the absence of Congo red, representing monomers.
The very intense second maximum is observed at smaller angles,
representing a specific large oligomer.
-lactoglobulin oligomer formed in the presence of 1 mM
Congo red. The relationship between RS and
Rg is quite sensitive to the shape and compactness
of a protein (see "Materials and Methods"). For an ideal spherical
particle RS/Rg = 1.29, whereas
for globular proteins the average value of this ratio is about 1.25 (25). The Congo red-bound oligomeric form of
-lactoglobulin has
RS/Rg = 1.26, consistent with a spherical shape, rather than a linear polymer.
structure is not a requirement for Congo red binding, since
cytoskeletal proteins have
,
, or coiled-coil structures. Our
data showing Congo red binding to proteins from different secondary
structure classes helps explain the false-positive results obtained due
to Congo red binding to cytoskeletal proteins. We suggest that caution
is necessary when using Congo red as a method for testing for fibrils
formed in vitro. Other dyes, such as thioflavin T, that are
more specific to amyloid fibrils and do not bind native proteins, are
better alternatives (46) to Congo red for in vitro detection
of fibrils (47).
(7), amylin (48), prions (49), and insulin),
there is considerable uncertainty as to the mechanism in which CR
interacts with fibrils. In several cases, evidence in support of an
electrostatic interaction has been reported (7, 51, 52),
however, in other cases it appears that specific interactions and not
simple electrostatic interactions are involved (27, 50, 53). The
necessity for planarity has been shown for the interaction with prion
fibrils (53). Since our results show that Congo red binds to native and
partially folded conformations of proteins, a likely mechanism of
inhibition of amyloid fibril formation is that Congo red preferentially
binds to the native or partially folded states and stabilizes them
(possibly as oligomers), thereby preventing formation of fibrils.
-sheets,
due to the regular spacing of both assemblies. However, our results
clearly show that this cannot be correct, since binding of CR to
non-
-sheet proteins occurs, and, in fact, Congo red binds to native
proteins from a wide variety of secondary structure classes. This
suggests that specific secondary structural elements are not a
requirement for binding of Congo red to proteins and amyloid fibrils.
We have demonstrated, for the first time, oligomerization of proteins
upon binding to Congo red, indicating that intercalation of dye
molecules between multiple protein molecules leading to large oligomers
is a probable mechanism of binding of Congo red to most native
proteins. The shape of these oligomers was determined to be relatively
spherical, indicating that the oligomerization does not lead to linear
complexes. It is likely that Congo red, an elongated sulfonated
hydrophobic molecule, binds to an exposed hydrophobic surface of the
native or partially folded conformations, probably with specific
complementary electrostatic interactions between charged side chains
and the sulfonate and amino groups of the dye, and induces
association without concomitant structural changes. The complex of
several protein molecules linked by intercalated Congo red molecules
may remain as a soluble oligomer, as for IL-2 and
-lactoglobulin, or
may become so large that it precipitates out of solution, as observed
for lysozyme. It is most likely that both the hydrophobic and the
electrostatic components of the structure of CR are critical for its
binding to proteins.
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ACKNOWLEDGEMENTS |
---|
We thank Jim Lewis, Keith A. Oberg, and
Pierre Souillac for helpful discussions. The SMA plasmid was a gift
from Fred Stevens, and insulin was provided by Novo Nordisk. The
-helical proteins were generous gifts from Drs. Jonathan King, Fran
Jurnak, and Steve Roderick. We thank Sangita Seshadri for preparing the apomyoglobin.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Chemistry and
Biochemistry, University of California, Santa Cruz, CA 95064. Tel.:
831-459-2744; Fax: 831-459-2935; E-mail: enzyme@cats.ucsc.edu.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M011499200
2 R. Khurana, K. A. Oberg, S. Sheshadri, L. Shi, and A. L. Fink, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CR, Congo red; CD, circular dichroism; DTSSP, 3,3'-dithiobis(sulfosuccinimidylpropionate); DSP, dithiobis(succinimidylpropionate); SMA, initials of the patient with light chain amyloidosis whose sequence information was used to generate synthetic recombinant proteins; ATR, attenuated total reflectance; FTIR, Fourier transform infrared spectroscopy; PAGE, polyacrylamide gel electrophoresis; SAXS, small angle x-ray scattering; IL-2, interleukin-2; RS, Stokes radius.
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