Co-incorporation of Ab40 and Ab42 to form mixed pre-fibrillar
aggregates
David Frost
1
, Paul M. Gorman
1
, Christopher M. Yip
2
and Avijit Chakrabartty
1
1
Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, and
2
Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering,
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
Senile plaques, the invariable hallmark and likely proximal
cause of Alzheimer’s disease (AD), are structured deposi-
tions of the 40- and 42-residue forms of the Ab peptide.
Conversely, diffuse plaques, which are not associated with
neurodegeneration, consist mainly of unstructured Ab42.
We have investigated the interaction between Ab40 and
Ab42 through an assay, which involves labeling both vari-
ants with an environment-sensitive fluorophore. We have
monitored association of Ab without fibrillar seeds, which
allows investigation of molecular species preceding fibrils.
Immediately upon mixture, Ab40 and Ab42 associate into
mixed aggregates, in which the peptides are unstructured and
relatively accessible to water. When left to incubate for an
extended period, larger, more tightly packed aggregates,
which show secondary structure, replace the small,
unstructured aggregates formed earlier. Our results show

pathology. Upon postmortem examination of AD brains,
senile plaques are invariably found in the limbic and
association cortices, surrounded by dead or dying
neurons, as well as activated microglia and reactive
astrocytes [1]. In several forms of familial AD, mutations
in the APP gene have been identified [9,10]. Also,
mutations of the presenilin genes have been linked to
familial AD, and appear to lead to an increase in the
ratio of Ab42 to Ab40 [11]. Transgenic mice over
expressing a mutant form of APP develop neurohistolo-
gical characteristics similar to those of AD patients
[12–14]. Perhaps most convincingly, Down’s syndrome
patients, who receive a triple dose of the genes present on
chromosome 21, including the APP gene, often show
senile plaque deposition and classical AD neurohistology
in their late 20s or early 30s, followed by progressive
cognitive and behavioral dysfunction in their mid 30s [15].
Unlike senile plaques, diffuse plaques are more loosely
packed depositions of mostly unstructured Ab42 [1].
Diffuse plaques are not associated with dead or dying
neurons, and have been found upon post mortem exami-
nation of the brains of elderly people who had not
exhibited AD symptoms [16–20]. Diffuse plaques are also
referred to as Ôpreamyloid plaquesÕ because of several lines
of evidence that point to them as precursors to senile
plaques. In the Down’s syndrome patients discussed earlier,
diffuse plaques are observed as early as age 12 years [21].
Similarly, mice transgenic for mutant human APP also
develop diffuse Ab42 plaques before fibrillar plaques
surrounded by dead and dying neurons [12–14].

species.
We believe that the approach of studying fibrillogenesis in
the context of Ab40 and Ab42 mixtures is advantageous.
Studies are usually of either Ab40 or Ab42, while it is known
that in vivo, both Ab40 and Ab42 are present, and their
interaction may play a key role in the transition between
relatively innocuous diffuse plaques and possibly neurotoxic
senile plaques. Furthermore, many in vitro studies examine
fibril formation without rigorously removing all fibril seeds,
thereby making it impossible to characterize all species
preceding fibrils. In the present study, we ensure a homo-
geneous starting solution of monomeric Ab peptides,
thereby permitting an examination of the interaction
between Ab40 and Ab42 throughout the fibrillogenic
pathway.
Materials and methods
Peptide synthesis
A PerSeptive Biosystems 9050 Plus peptide synthesizer
was used to separately prepare both Ab40 and Ab42 by
solid phase peptide synthesis. An active ester coupling
procedure, employing O-(7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate of 9-fluorenyl-
methoxycarbonyl amino acids was used. The magnitude
of the syntheses was 0.05 mmol, and a three times excess
of reagents was used. The peptides were cleaved from the
resin with 95 : 5 trifluoroacetic acid and anisol mixture.
The cleavage mixture was incubated at room temperature
for 30 min, and the resin removed by filtration. Bromo-
trimethylsilane was added to a final concentration of
12.5% (v/v). The peptides were then precipitated and

revealed the major peptide present in each synthesis to be
the correct labeled Ab, with a minor contaminant of
unlabeled Ab.
Preparation of stock peptide solutions without fibril
seeds
After chromatographic separation as described above, both
the labeled and unlabeled peptides were separately stored
at pH 10 at 4 °C until use. This method of stock storage
has previously been used by us [25] and others [29] to
successfully prevent the formation of fibril seeds.
Peptide concentration determination
For unlabeled Ab peptides, tyrosine absorbance of UV
light (275 nm) was used to determine concentration
in 0.15% NH
4
OH by Beer–Lambert law (e ¼ 1390
cm
)1
Æ
M
)1
[30]). Each concentration obtained was multi-
plied by the appropriate dilution factor to obtain stock
concentrations. The EDANS-labeled peptide stock con-
centration was determined by EDANS absorbance at
338 nm (e ¼ 6500 cm
)1
Æ
M
)1

M
for
EDANS-Ab42, both stored at pH 10, 4 °C. All solutions
tested that included labeled Ab had a concentration of
0.1 l
M
EDANS-Ab. Therefore, for the solutions with
Ó FEBS 2003 Pre-fibrillary association between Ab40 and Ab42 (Eur. J. Biochem. 270) 655
EDANS-Ab40, 5.8 lL of stock was added. Similarly, for
solutions with EDANS-Ab42, 1.6 lL of stock was added.
Deionized water was then added to each sample, such
that the final volume was 300 lL. A 100-m
M
phosphate-
buffered solution was prepared, and separated into
aliquots, with pHs ranging from 6.8 to 7.0 in increments
of 0.05 pH units. Two hundred microliters of the buffer
with appropriate pH to make the final pH near 7.0 (i.e.
compensate for the addition of pH 10 Ab stocks) was
added, and 0.1 m
M
NaOH or HCl was added to make
the pH exactly 7.0, just prior to measurement. In most
cases the final adjustment involved no more than 1–2 lL
of acid or base, and therefore had negligible effect on the
final volume. This pH adjustment just prior to measure-
ment ensures that the fibrillogenesis process does not start
before measurements are taken. Controls (i.e. unlabeled
Ab alone and EDANS-Ab + hen lysozyme) were pre-
pared exactly as above, with the exception that the hen

correct for the effect of light scattering by large aggregates.
The resultant spectrum was then integrated over the
wavelengths of 400–550 nm. In order to correct for daily
variation in the UV lamp and slight variations in bandpass,
as well as the minor unlabeled contaminant of EDANS-Ab
stocks described above, the fluorescence of EDANS-Ab
alone was subtracted from all other measurements, giving a
normalized measure of the fluorescence of EDANS-Ab at
different concentrations over time. All fluorescence experi-
ments were conducted in three different trials on different
days.
Circular dichroism spectroscopy
CD spectra were recorded on an Aviv Circular Dichroism
Spectrometer model 62DS at 25 °C. Spectra were obtained
from 190 to 260 nm (1 mm path length, 1 nm step size,
1 nm bandwidth).
Atomic force microscopy
All solution tapping atomic force microscopy images
were acquired using a combination contact/tapping mode
liquid cell fitted to a Digital Instruments Nanoscope IIIA
MultiMode scanning probe (Digital Instruments, Santa
Barbara, CA, USA). The AFM images were acquired
using the E scanning head, which has a maximum lateral
scan area of 14.6 · 14.6 lm. Samples were made by
diluting the appropriate Ab stocks with 100 m
M
phosphate buffer (pH 7). Five microliters of the mixed
sample solution were transferred onto a freshly cleaved
mica surface, and the sample was sealed in the liquid cell.
Sizes and volumes were calculated using Digital Instru-

combination of Ab40 and Ab42 heterogeneous associ-
ation, as well as homogeneous association was examined.
Given that the threshold concentration for fibril forma-
tion of Ab40 at neutral pH is between 10 and 40 l
M
[27],
0, 10, 20 and 30 l
M
Ab are ideal concentrations to
monitor prefibrillar species.
To start the fibrillogenesis process, the pH of the
solution is lowered from 10 to 7 by addition of
phosphate buffer. The AEDANS fluorophore absorbs
at approximately 350 nm, and emits at approximately
480 nm. In samples with only AED-Ab, fluorescence at
480 nm is relatively low due to fluorescence quenching
by water (Fig. 1). As the label is sequestered by
unlabeled Ab, fluorescence increases. In order to control
for light scattering by the peptides as an explanation for
increased fluorescence readings, we also scanned 10, 20
and 30 l
M
unlabeled Ab over the same wavelengths, and
subtracted these spectra from the corresponding ones
with EDANS-Ab. In Fig. 3B, we show the unsubtracted
fluorescence for EDANS-Ab40 incorporating into
unlabeled Ab40 at the early time period, as well as the
subtracted fluorescence and the difference, over the three
concentrations tested. As a second control, we prepared
samples of EDANS-Ab40 or EDANS-Ab42 with 0, 10,

pH (Fig. 3). Addition of labeled Ab40 to increasing
concentrations of both unlabeled Ab40 or unlabeled
Ab42 resulted in an increase in fluorescence intensity
indicating that labeled Ab40 incorporates into both
aggregates of unlabeled Ab40 and Ab42. Similarly,
labeled Ab42 was found to incorporate into both
aggregates of unlabeled Ab40 and Ab42 (Fig. 3C).
Significantly, the observed incorporation is Ab-specific;
controls of EDANS-Ab mixed with the same concentra-
tions of hen lysozyme showed negligible incorporation.
Three trials were conducted on separate days, and yielded
these results consistently. The observed incorporation is
not due to light scattering from increasing protein
concentrations, as spectra of unlabeled peptide alone
were subtracted from their counterparts with EDANS-
labeled Ab to generate the data shown in Fig. 3. All
EDANS peaks in early spectra (i.e. upon mixture) occur
around the known maximum of approximately 480 nm
(Fig. 4).
CD spectroscopy at early time period
Immediately upon mixing, at the time point when
association between Ab species occurs and aggregates
are small and amorphous, CD shows a spectrum of a
random coil or unstructured conformation (data not
shown). The spectrum shows a minimum at approxi-
mately 190 nm. The presence of small aggregates in these
samples can confound the interpretation of CD spectra.
However, we are confident that light-scattering effects
have not adversely influenced the results because the
spectrum so closely resembles that of a typical random

100000
150000
200000
250000
300000
350000
0 5 10 15 20 25 30 35
[Unlabeled peptide] (µM)
[Unlabeled peptide] (µM)
Fluorescence (400-550nm)
Fluorescence (400-550nm)
0
50000
100000
150000
200000
250000
300000
350000
400000
10 20 30
Fluorescence (400-550nm)
[Unlabeled peptide] ( M)
A
B
C
Fig. 3. Fluorescence of EDANS-Ab40 with unlabeled Ab40, Ab42 or
hen lysozyme immediately after mixing. (A) Fluorescence of 0.1 l
M
EDANS-Ab40 with 0, 10, 20 and 30 l

Significantly, the EDANS peak of approximately
480 nm shifts to approximately 420 nm, concomitant
with late aggregate formation. In addition, the magnitude
of EDANS fluorescence is approximately five- to 10-fold
higher with late aggregates relative to early aggregates,
indicating that the fluorophore is more sequestered in the
late aggregate. We have consistently observed that
structured aggregate formation is accompanied by a blue
shift and increased intensity in the EDANS spectrum.
Figure 7 demonstrates that a biphasic distribution of
EDANS fluorescence exists at certain Ab concentrations,
indicating a mixture of unstructured and structured Ab
aggregates in the solution. All fluorescence scans of
EDANS-Ab alone show maxima at 480 nm, indicating
no structured aggregate formation in these samples, as
expected by the trace concentration of labeled Ab (i.e.
0.1 l
M
). The unshifted spectrum of AEDANS-Ab alone
after extended incubation (Fig. 7) also eliminates the
possibility that the behavior of the EDANS fluorophore
changes due to the incubation itself rather than a change
in the aggregate species. As mentioned above, three trials
were conducted over separate days, and yielded similar
results.
CD spectroscopy after extended incubation
CD spectra of samples showing large, structured aggre-
gates and blue-shifted EDANS fluorescence were taken.
As mentioned above, large aggregates can confound CD
data, but the spectrum obtained shows definite secondary

M
unlabeled Ab40 alone subtracted. Peaks occur
at normal EDANS fluorescence maximum
of approximately 480 nm. (B) Fluorescence
spectrum of 0.1 l
M
EDANS-Ab40 with 0 (h),
10 (j),20(s) and 30 (d) l
M
unlabeled
Ab42 immediately upon mixing. Spectra of 0,
10, 20 and 30 l
M
unlabeled Ab42 alone
subtracted. Peaks occur at normal EDANS
fluorescence maximum of approximately
480 nm.
Ó FEBS 2003 Pre-fibrillary association between Ab40 and Ab42 (Eur. J. Biochem. 270) 659
because the EDANS spectrum shifts to a 420-nm peak when
structured aggregates are present. Hence, we are able to
detect the formation of structured aggregates after an
extended incubation period (3 months).
There is evidence to suggest that the aggregates formed
immediately upon lowering the pH are similar to the
diffuse plaques observed in vivo. The aggregates are
morphologically unstructured, and form a diffuse lawn
A
B
Fig. 5. AFM images of Ab40 (A) and hen
lysozyme (B) after extended incubation.

of typical Ab amyloid fibrils. Fluorescence shows that the
peptides are highly sequestered from water, indicating
tighter packing. The fluorescence spectrum shifts to
420 nm from 480 nm, indicating a significant change in
fluorophore behavior. Finally, CD shows the aggregates
to be structured, with spectra similar to those of b-sheet
(Ab fibrils found in senile plaques also have b-sheet
secondary structure).
After 3 months of incubation, fibrils were not detected
by EM or AFM in any of the 0, 10, 20 and 30 l
M
Ab
samples tested. Because we undertook this study to
examine the early aggregation events, not the fibrils per
se,wehavechosenAb concentrations near or below the
known threshold for Ab40 fibril formation under the
conditions tested. It is therefore not surprising that
fibrils have not formed in these samples. It is also
important to note that fibril formation is quite difficult
to achieve de novo. As described in the Materials and
methods section, we have employed a rigorous proce-
dure to prevent the formation of fibrillar seeds in our
stock Ab solutions. This allows us to examine prefibril-
lar structures. We are confident therefore that by
examining concentrations at or near threshold for fibril
formation, prefibrillar structures are the major species
present.
After sufficient time for structured aggregates to form,
we find that both homogeneous and mixed aggregates
have formed, but that Ab40 shows a slight bias towards

plaques.
This work has demonstrated the possibility for Ab to
form both mixed early unstructured aggregates (similar to
diffuse plaques) and late structured aggregates (possibly
an intermediate in the transition to senile plaques), and
has shown that, in vitro, Ab40 and Ab42 associate early
in the fibrillogenesis pathway. We have also demonstra-
ted an interesting property of the EDANS fluorophore,
namely that its fluorescence spectrum shifts concomitant
with structured aggregate formation. This could be quite
useful in other fibrillogenesis studies. More work is
needed to elucidate not only the aggregation and
fibrillogenesis pathway of Ab40, which is an area of
much active research, but also the role that Ab42/Ab40
interaction plays in the formation of senile plaques. This
study provides a starting point for further investigation in
this regard.
Fig. 6. Spectra from samples excited at 350 nm and scanned from 360 to
600 nm. Samples were excited at 350 nm and scanned from 360 to
600 nm. The resultant spectra were integrated over 400–550 nm. Scans
of 0, 10, 20 and 30 l
M
unlabeled peptide alone over the same wave-
lengths were subtracted from the EDANS spectra obtained. (A)
Fluorescence of 0.1 l
M
EDANS-Ab40 with 0, 10, 20 and 30 l
M
unlabeled Ab40 (d),Ab42 (s) or hen lysozyme (j) after incubation
for approximately 3 months at pH 7. (B) Fluorescence of 0.1 l

5. Busciglio, J., Gabuzda, D.H., Matsudaira, P. & Yankner, B.A.
(1993) Generation of beta-amyloid in the secretory pathway in
neuronal and nonneuronal cells. Proc. Natl Acad. Sci. USA 90,
2092–2096.
6. Haass, C., Schlossmacher, M.G., Hung, A.Y., Vigo-Pelfrey, C.,
Mellon, A., Ostaszewski, B.L., Lieberburg, I., Koo, E.H., Schenk,
D., Teplow, D.B. & Selkoe, D.J. (1992) Amyloid b-protein is
produced by cultured cells during normal metabolism. Nature 359,
322–325.
7. Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H.,
Sinha, S., Schlossmacher, M.G., Whaley, J., Swindlehurst, C.,
McCormack, R., Wolfert, R., Selkoe, D.J., Lieberburg, I.
& Schenk, D. (1992) Isolation and quantification of
soluble Alzheimer’s b-peptide from biological fluids. Nature 359,
325–327.
8.Shoji,M.,Golde,T.E.,Ghiso,J.,Cheung,T.T.,Estus,S.,
Shaffer, L.M., Cai, X.D., McKay, D.M., Tintner, R.,
Frangione, B. & Younkin, S.G. (1992) Production of the
Alzheimer amyloid b protein by normal proteolytic processing.
Science 258, 126–129.
9. Goate, A., Chartier-Harlin, M.C., Mullan, M., Brown, J., Craw-
ford,F.,Fidani,L.,Giuffra,L.,Haynes,A.,Irving,N.,James,L.,
Mant, R., Newton, P., Rooke, K., Roques, P. & Talbot, C. (1991)
Segregation of a missense mutation in the amyloid precursor
protein gene with familial Alzheimer’s disease. Nature 349, 704–
706.
10. Murrell, J., Farlow, M., Ghetti, B. & Benson, M.D. (1991) A
mutation in the amyloid precursor protein associated with here-
ditary Alzheimer’s disease. Science 254, 97–99.
11. Suzuki,N.,Iwatsubo,T.,Odaka,A.,Ishibashi,Y.,Kitada,C.&

M
unlabeled Ab40
after incubation for approximately 3 months.
Spectra of similarly incubated 0, 10, 20 and
30 l
M
unlabeled Ab40 alone subtracted. Peak
occurs at shifted EDANS fluorescence
maximum of approximately 420 nm, which
corresponds to structured aggregate forma-
tion. A somewhat biphasic distribution of
maxima (420 and 480 nm) is visible in the
10 l
M
spectrum, corresponding to a mixed
population of structured and unstructured
aggregates. (B) Fluorescence spectrum of
0.1 l
M
EDANS-Ab40 with 0 (h),10(j),20
(s) and 30 (d) l
M
unlabeled Ab42 after
incubation for 3 months. Spectra of similarly
incubated 0, 10, 20 and 30 l
M
Ab42 subtrac-
ted. Biphasic distribution of EDANS peaks
corresponds to a mixed population of struc-
tured and unstructured aggregates.

19. Joachim, C.L., Morris, J.H. & Selkoe, D.J. (1989) Diffuse senile
plaques occur commonly in the cerebellum in Alzheimer’s disease.
Am. J. Pathol. 135, 309–319.
20. Yamaguchi, H., Nakazato, Y., Hirai, S., Shoji, M. & Harigaya, Y.
(1989) Electron micrograph of diffuse plaques. Initial stage of
senile plaque formation in the Alzheimer brain. Am. J. Pathol. 135,
593–597.
21. Lemere, C.A., Blustzjan, J.K., Yamaguchi, H., Wisniewski, T.,
Saido, T.C. & Selkoe, D.J. (1996) Sequence of deposition of het-
erogeneous amyloid beta-peptides and APO E in Down syn-
drome: implications for initial events in amyloid plaque formation.
Neurobiol. Dis. 3, 16–32.
22. Hardy, J. (1997) Amyloid, the presenilins and Alzheimer’s disease.
Trends Neurosci. 20, 154–159.
23. Hasegawa, K., Yamaguchi, I., Omata, S., Gejyo, F. & Naiki, H.
(1999) Interaction between A beta (1–42) and A beta (1–40) in
Alzheimer’s beta-amyloid fibril formation in vitro. Biochemistry
38, 15514–15521.
24. Walsh, D.M., Klyubin, I., Fadeeva, J.V., Cullen, W.K., Anwyl,
R., Wolfe, M.S., Rowan, M.J. & Selkoe, D.J. (2002) Naturally
secreted oligomers of amyloid beta protein potently inhibit hip-
pocampal long-term potentiation in vivo. Nature 416, 535–539.
25. Huang, T.H., Yang, D.S., Plaskos, N.P., Go, S., Yip, C.M.,
Fraser, P.E. & Chakrabartty, A. (2000) Structural studies of
soluble oligomers of the Alzheimer beta-amyloid peptide. J. Mol.
Biol. 297, 73–87.
26. Stine, W.B. Jr, Snyder, S.W., Ladror, U.S., Wade, W.S., Miller,
M.F., Perun, T.J., Holzman, T.F. & Krafft, G.A. (1996) The
nanometer-scale structure of amyloid-beta visualized by atomic
force microscopy. J. Protein Chem. 15, 193–203.

35. Suzuki, N., Cheung, T.T., Cai, X.D., Odaka, A., Otvos, L.,
Eckman, C., Golde, T.E. & Younkin, S.G. (1994) An increased
percentage of long amyloid beta protein secreted by familial
amyloid beta protein precursor (beta APP717) mutants. Science
264, 1336–1340.
Ó FEBS 2003 Pre-fibrillary association between Ab40 and Ab42 (Eur. J. Biochem. 270) 663