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ANALYTICAL
Analytical Biochemistry 329 (2004) 238–246
BIOCHEMISTRY
www.elsevier.com/locate/yabio
Electron microscopy as a quantitative method for investigatingtau fibrillization
Mihaela Neculaa and Jeff Kuretb,*
a Biophysics Program, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USAb Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USA
Received 4 December 2003
Available online 30 April 2004
Abstract
Fibrillization of tau protein is a hallmark lesion in Alzheimer�s disease. To clarify the utility of electronmicroscopy as a quantitative
assay for tau fibrillization in vitro, the interaction between synthetic tau filaments and carbon/formvar-coated grids was characterized
in detail. Filament adsorption onto grids was hyperbolic when analyzed as a function of time or bulk protein concentration, with no
evidence for competitive displacement or elution from other components in the reaction mixture. Filament length measurements were
linear with filament concentration so long as the concentration of total tau protein in the sample was held constant, suggesting that
measurement of filament lengths was accurate under these conditions. Furthermore, exponential filament length distributions were not
significantly affected by adsorption time or filament concentration, suggesting that preferential binding among filaments of differing
lengths was minimal. However, monomeric tau protein was found to be a strong competitor of filament adsorption, indicating that
comparison of filament length measurements at different bulk tau concentrations should be interpreted with caution.
� 2004 Elsevier Inc. All rights reserved.
Keywords: Tau; Amyloid; Fibrillization; Adsorption; Interfacial concentration; Microscopy
Alzheimer�s disease (AD)1 is characterized in part by
the aggregation of tau protein into neurofibrillary le-
sions [1]. Although amorphous aggregation dominates
early stages of disease [2,3], cognitive decline correlates
with the fibrillization of tau into ordered filaments
containing cross b-sheet structure [2,4]. Therefore,characterization of the fibrillization reaction is of central
importance for clarifying the underlying events in
tauopathic neurodegenerative diseases such as AD.
Quantitative assays for fibrillization reactions are
limited but include dye-based fluorescence spectroscopy
[5,6], static and dynamic light scattering [7,8], and sed-
imentation [9], each of which has specific limitations and
weaknesses [10]. Above all, none of these solution-based
* Corresponding author. Fax: 1-614-292-5379.
E-mail address: kuret.3@osu.edu (J. Kuret).1 Abbreviations used: AD, Alzheimer�s disease; AA, arachidonic
acid; N774, (2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiaz-
ole); EM, electron microscopy, PHF, paired helical filament; DMSO,
dimethyl sulfoxide.
0003-2697/$ - see front matter � 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2004.02.023
methods involve direct visualization of fibrils, which is
critical for assessing filament morphology and length
distributions. Thus electron microscopy has emerged as
an essential adjunct to solution methods for character-
ization of tau fibrillization [11]. But the utility of the
method for rigorous quantitation has been questioned.On the one hand, experience with authentic filament
preparations suggests that filament lengths estimated by
electron microscopy are linear with filament concentra-
tion and therefore reliable [12]. On the other hand it has
been claimed on the basis of experience with synthetic
filaments prepared with heparin that quantitation of fi-
bril lengths using electron microscopy is problematic
[13,14].We employ electron microscopy as a quantitative
assay for synthetic tau filaments formed from re-
combinant tau protein and fatty acid inducer [9,11].
Fibrillization occurs in solution, as with other methods,
but resultant filaments are detected only after
adsorption of reaction products onto the hydrophobic
surface of grids coated with formvar (a polyvinyl formal
M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246 239
polymer) and carbon. Thus adsorption phenomena havea large influence on the accuracy of filament length
measurements by this method. Adsorption of proteins
onto various surfaces has been studied for many
proteins, including fibrinogen [15], wheat proteins [16],
serum albumin [17], lysosyme [18], insulin [19], and
b-lactoglobulin [20]. From such experiments it was
shown that adsorption depends upon temperature, pH,
ionic strength, and protein concentration [21–25]. Inaddition, adsorption from protein mixtures can be a
competitive and dynamic process, where initially ad-
sorbed proteins can undergo conformational changes
and displace other proteins in a time- and concentra-
tion-dependent manner [15,26,27]. The later process is
known as the Vroman effect, and it can produce non-
linearity in adsorption-based assays [28,29].
Here we characterize the interaction of tau fibrilliza-tion reaction products with formvar/carbon-coated grids
as a function of protein concentration and adsorption
time. In addition, we investigate the effect of soluble tau
protein, fatty acid, and tau fibrillization inhibitor N744
on fibril–grid surface interaction. Results show the
conditions under which electron microscopy can be used
to give accurate estimates of tau filament length.
Materials and methods
Materials
Recombinant double mutant htau40C291A;C322A and
htau37 were expressed and purified as described previ-
ously [8,30,31]. AA (Fluka, Milwaukee, WI) was dis-solved in 100% ethanol and stored under argon gas at
)80 �C until used. Tau polymerization inhibitor N744
was the generous gift of Neuronautics (Evanston, IL)
and was dissolved and stored at )20 �C in DMSO.
Formvar/carbon-coated grids (300-mesh), glutaralde-
hyde, and uranyl acetate were from Electron Micros-
copy Sciences (Ft. Washington, PA). Carboxylate-
conjugated polystyrene microspheres (90 nm diameter;molecular area¼ 12�A2/eq) were obtained from Bangs
Laboratories (Fishers, IN).
Tau fibrillization
Tau isoforms htau40C291A;C322A or htau37 (4–10 lM)
were incubated without agitation in Assembly Buffer
(10mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonicacid, pH 7.4, 100mM NaCl, and 5mM dithiothreitol)
at 37 �C for up to 24 h in the presence of either AA
(0–200 lM) or carboxylate-substituted polystyrene
microspheres (124 pM). Reactions were terminated with
glutaraldehyde treatment (2% final concentration) for
5–30min, diluted 0- to 40-fold in Dilution Buffer
(Assembly Buffer containing 2% glutaraldehyde) alone
or containing various additions, and subjected toelectron microscopy assay as described below. When
present, tau fibrillization inhibitor N744 was accompa-
nied by DMSO vehicle, which was limited to 1% in all
reactions.
Transmission electron microscopy
Aliquots (50 ll) of fixed reactions were adsorbed(0.25–15min) onto 300-mesh formvar/carbon-coated
copper grids. The resultant grids were washed with
water, stained (1min) with 2% uranyl acetate, washed
again with water, blotted dry, and viewed in a Phillips
CM 12 microscope operated at 65 kV. Random images
from each experimental condition were captured on film
at 8000- to 22,000-fold magnification, digitized, cali-
brated, and imported into Optimas 6.5.1 for quantita-tion of filament length and number as described
previously [30]. Interfacial filament concentration (Cf ) is
defined as the summed lengths of all filaments >50 nm in
length per unit area and is reported in units of nm/
lm2 � SD as calculated from three to six random images
per condition. Interfacial microsphere concentration
(CM) is defined as the number of microspheres per unit
area and is reported in units of microspheres/lm2 � SDas calculated from three to six random images per
condition.
Analytical methods
Hyperbolic adsorption data were fit to the Toth iso-
therm [32],
C ¼ Cmax
ax
ð1þ ðaxÞbÞ1=b;
where C is the interfacial concentration measured at
bulk concentration x, Cmax is the interfacial concentra-
tion at saturation, a is a constant, and b is the Toth
coefficient reported� SE without units.
Filament length distributions were fit to the expo-
nential equation,
y ¼ aebx;
where y is the percentage of all filaments filling a bin of
length interval x and b is a constant reported in units oflength�1 � SE.
Results of linear regression analysis are reported�SE.
Results
Effect of dilution on filament adsorption
Experimentation began with mutant tau con-
struct htau40C291A;C322A because it simplifies and speeds
240 M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246
filament quantitation [8]. Adsorption of filamentoushtau40C291A;C322A onto formvar/carbon-coated EM grids
was examined as a function of bulk tau concentration
after serial dilution of glutaraldehyde-fixed fibrillization
products into Dilution Buffer at room temperature.
Adsorption times of 1 and 15min were studied. Resul-
tant plots were linear up to 1 lM total tau concentration
for 1-min adsorption and up to 0.5 lM tau for 15-min
adsorption, with initial slopes dependent on adsorptiontime (Fig. 1). Above these tau concentrations, isotherms
abruptly became nonlinear and appeared to approach
saturation. There was no indication of decreasing fila-
ment adsorption with decreasing dilution, suggesting
that adsorbed fibrils were not displaced or eluted by
other components of the assembly system such as mo-
nomeric tau or AA (i.e., there was no Vroman effect).
Extended binding linearity followed by abrupt satu-ration is characteristic of type C2 adsorption behavior in
the empirical Giles et al. [33] classification system.
Therefore, the Toth isotherm was used to fit these data
because it accurately models linearity at low sorbate
concentrations [34]. Saturation as estimated by extrap-
olation from the resultant isotherms was 3150� 80
(Toth coefficient¼ 4.1� 1.1) and 3510� 40 (Toth coef-
ficient¼ 4.5� 1.2) nm/lm2 length for 1- and 15-minadsorption times, respectively. Assuming a mass per unit
length of 75� 17 kDa/nm [35] yielded molecular areas of
15–21 cm2/lg filamentous tau protein at saturation.
These values are below typical values determined for
globular proteins in single-component experiments [36].
Fig. 1. Tau filament adsorption isotherm. Filaments prepared (3 h at
37 �C) from recombinant double mutant htau40C291A;C322A (4lM) in
the presence of AA inducer (75lM) were fixed in 2% glutaraldehyde,
serially diluted in Dilution Buffer, adsorbed onto carbon/formvar-
coated grids for either 1min (j) or 15min (�), and then viewed by
transmission electron microscopy after negative staining. Each point
represents Cf (the interfacial concentration of filaments >50 nm in
length) as a function of dilution (where a dilution of unity, 4lM total
tau), whereas each curve represents the best fit of data points to the
Toth isotherm. Under these conditions, adsorption increased linearly
with concentration until an abrupt break point (type C2 behavior in
the nomenclature of Giles et al. [33]).
These data suggest that filament adsorption ontocarbon-coated grids is linear only up to a threshold
concentration related to saturation on the grid surface
and that filament length measurements made above this
level are not proportional to filament concentrations in
solution.
Adsorption timecourse
To investigate the kinetics of filament adsorption,
htau40C291A;C322A filaments were prepared from 4 lMtotal tau and adsorbed onto grids neat or diluted 5-fold
in Dilution Buffer. Neat reaction product adsorbed and
saturated within 15 s, with interfacial filament concen-
tration remaining constant over the entire 15-min time
course (Fig. 2). Diluted reaction products adsorbed
more slowly with hyperbolic kinetics (Fig. 2). Bothcurves approached saturation monotonically without
evidence of transient adsorption (a manifestation of the
Vroman effect). These data suggest that adsorption time
is an important variable in measurement of filament
lengths and therefore samples investigated by EM
should be compared only if they were adsorbed onto
grids for identical periods of time.
Interfering substances
In addition to filaments, fibrillization reactions con-
tain fatty acid and unincorporated tau protein, each of
which could potentially compete with filament binding
and contribute to molecular crowding at the grid
Fig. 2. Kinetics of tau filament adsorption onto grids. Filaments
prepared (3 h at 37 �C) from recombinant double mutant
htau40C291A;C322A (4lM) in the presence of AA inducer (75lM) were
fixed in 2% glutaraldehyde and serially diluted in Dilution Buffer. The
adsorption of undiluted fibrils (�) and 5-fold-diluted fibrils (j) onto
carbon/formvar-coated grids was then measured as a function of ad-
sorption time at room temperature. Each point represents Cf as a
function of adsorption time, whereas each line represents the best fit of
the data points to a rectangular hyperbola. Although undiluted reac-
tion products adsorbed within 15 s, no time-dependent decrease in Cf
was observed under these conditions.
M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246 241
surface. In fact these may account for the relatively highmolecular areas associated with filament saturation.
Moreover, both proteins and anionic surfactants can
elute adsorbed proteins when present at high concen-
trations and therefore complicate quantitation [37]. To
determine which components of the reaction mixture
contributed to crowding, filaments synthesized from
4 lM htau40C291A;C322A were diluted 5- to 10-fold in
Dilution Buffer containing various concentrations ofAA or monomeric tau and analyzed by electron mi-
croscopy after 1- or 15-min adsorption times. Results
showed that AA concentrations up to at least 50 lM did
not compete for filament adsorption regardless of dilu-
tion or incubation time (Fig. 3A). In fact, concentra-
tions up to 200 lM appeared to be without effect,
although samples adsorbed 1min containing >100 lMAA or adsorbed 15min containing >50 lM AA werenot quantified due to the fibril clumping that occurred in
this assay format. These data suggest that normal
working concentrations of anionic surfactant inducers
Fig. 3. Anionic surfactants do not interfere with filament adsorption.
Filaments prepared (3 h at 37 �C) from recombinant double mutant
htau40C291A;C322A (4lM) in the presence of AA inducer (75lM) were
fixed in 2% glutaraldehyde and diluted into Dilution Buffer containing
various concentrations of AA or small-molecule tau fibrillization in-
hibitor N744. Each point represents interfacial filament concentration
(Cf ) as a percentage of control adsorption conducted in the absence of
additions, whereas each line represents linear regression analysis of the
data points. (A) Filaments were either diluted 5-fold and adsorbed
1min (j) or diluted 10-fold and adsorbed 15min (�) in the presence of
varying AA concentrations. AA did not interfere with adsorption at
concentrations up to 100lM (although these concentrations led to
filament clumping in the 15-min format, thereby making quantitation
questionable). (B) Filaments were either diluted 5-fold and adsorbed
1min (d) or diluted 10-fold and adsorbed 15min (s) in the presence of
varying concentrations of N744. Under these diverse conditions, N744
at concentrations up to 50 lM did not interfere with filament ad-
sorption.
(0–100 lM) do not interfere with assay of tau fibrilliza-tion.
The ability of small-molecule agents to interfere with
adsorption also was examined using tau fibrillization
inhibitor N744, a member of the Congo red family of
dyes [38]. These compounds are thought to bind all
along the length of filaments containing cross b-sheetconformation [39]. Dilution of filaments into Dilution
Buffer containing up to 50 lM N744 did not affectfilament adsorption (Fig. 3B), suggesting that the
presence of small-molecule ligands such as N744 at
working concentrations (0–4 lM) does not affect assay
performance.
In contrast, the presence of monomeric tau protein in
Dilution Buffer was strongly competitive above 1 lM for
1-min adsorption and 0.4 lM for 15-min adsorption
(Fig. 4). These data suggest that bulk tau protein maycontribute to the saturation of interfacial filament con-
centration observed as a function of total tau concen-
tration and that measurement of tau fibrillization at
different tau concentrations can misstate true levels of
fibrillization.
Together these data indicate that fibrillization in the
presence of varying inducer and inhibitor concentrations
can be directly compared at constant tau concentration.However, measurements made in the presence of vary-
ing tau concentrations cannot be directly compared
unless diluted to linearity.
Fig. 4. Monomeric tau protein interferes with filament adsorption.
Filaments prepared (3 h at 37 �C) from recombinant double mutant
htau40C291A;C322A (4lM) in the presence of AA inducer (75lM) and
fixed in 2% glutaraldehyde were diluted 5-fold into Dilution Buffer
containing various concentrations of monomeric htau40C291A;C322A,
adsorbed onto carbon/formvar-coated grids for either 1min (j) or
15min (�), and then viewed by transmission electron microscopy after
negative staining. Each point represents interfacial filament concen-
tration (Cf ) as a percentage of control adsorption conducted in the
absence of additions, whereas each line is drawn to aid visualization
without being fit to a model. Monomeric tau protein competes with
filament adsorption at concentrations above 1lM in the 1-min and
0.4 lM tau in the 15-min adsorption paradigms.
242 M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246
Assay linearity at constant total protein concentration
The above data suggest that, despite the complexity
of reaction mixtures, adsorption behavior reduces to a
binary system (monomeric and filamentous tau popu-
lations) at constant temperature and buffer conditions.
These two components can directly compete for ad-
sorption, but the lack of visible Vroman effect in both
time course and dilution formats suggest that exchangeand elution reactions are minimal. Under these condi-
tions, adsorption of each component of a binary system
should reflect their relative bulk concentrations in so-
lution [15,40]. Therefore the interfacial binding of
filaments should be a linear function of filament con-
centration so long as the competing agent, monomeric
tau protein, is held constant and saturation is not
reached. To test this hypothesis, aliquots of filamentswere diluted up to 40-fold in Dilution Buffer containing
varying concentrations of tau such that the final total
tau concentration remained constant. Diluted samples
were then adsorbed onto grids (1-min incubation time)
and examined by transmission electron microscopy.
Two tau isoforms were used: htau40C291A;C322A at 4 lMbecause this four-repeat isoform fibrillizes efficiently at
this concentration and htau37 at 10 lM because three-repeat isoforms nucleate less efficiently and require
higher concentrations for reliable filament length mea-
surements [30]. The resultant plots of filament interfacial
concentration versus dilution were linear for both tau
isoforms (Fig. 5). Linearity was observed after 15-min
adsorption times also (data not shown). These data
Fig. 5. Filament adsorption is linear at constant total protein con-
centrations. Filaments prepared from 4 lM htau40C291A;C322A (3 h at
37 �C; 75 lM AA) or 10lM htau37 (24 h at 37 �C) in the presence of
AA inducer were fixed in 2% glutaraldehyde and then serially diluted
into Dilution Buffer containing various concentrations of monomeric
tau protein so that the final concentration of tau was held constant at
either 4lM (j, htau40C291A;C322A) or 10 lM (�, htau37). Each point
represents Cf as a function of dilution after 1-min adsorption, whereas
each line represents linear regression analysis of the data points. Fil-
ament adsorption is linear with filament concentration at constant
total tau concentration.
confirm linearity of filament length measurements in thepresence of constant total tau concentration and suggest
that length measurements are reliable below saturation
so long as this condition is met.
Filament length distributions
Although no displacement of filaments as a function
of time or tau concentration was observed, it is possiblethat filaments of differing lengths adsorb at different
rates, resulting in biased apparent length distributions.
To address this issue, the length distribution of filaments
prepared from 4 lM htau40C291A;C322A and 75 lM AA
was determined as a function of adsorption time and
dilution at constant total tau concentration. All length
distributions were exponential, consistent with the time-
dependent fibrillization pathway induced by AA (Fig. 6)and therefore were quantified using exponential slope
parameter b as described under Materials and methods.
Undiluted samples adsorbed for 1 or 15min had bvalues of )5.34� 0.67 and )5.02� 0.64 lm�1 (Fig. 6),
consistent with previously published results (b ¼ �5:4�0:2 lm�1 at 3 h; Fig. 1 in [9]). Similarly, the length dis-
tributions of samples diluted 4-fold and adsorbed for 1
or 15min had b values of )5.55� 0.71 and)5.83� 0.70 lm�1, although the number of filaments
adsorbed onto grids was 3- to 5-fold lower in these di-
luted samples (Fig. 6). These data, together with data
presented in Fig. 5, suggest that length distributions are
Fig. 6. Length distributions are independent of tau protein concen-
tration and adsorption time. Filaments formed from htau40C291A;C322A
incubated (3 h at 37 �C) in the presence of 75lM AA were fixed in 2%
glutaraldehyde and either diluted 4-fold into Dilution Buffer (j;�) or
used undiluted (d;s). All filament populations were then adsorbed
onto grids for either 1min (j;d) or 15min (�;s). The lengths of
filaments P 50 nm in length were then measured from digitized images
and plotted. Each data point represents the number of analyzed fila-
ments segregating into consecutive length intervals (100-nm bins),
whereas each line represents the best fit of data points to an expo-
nential regression. Length distributions remained exponential with
nearly identical slopes regardless of protein concentration or adsorp-
tion time.
M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246 243
not dependent on adsorption time when assayed atconstant total tau concentration.
Microsphere adsorption is tau protein dependent
Anionic microspheres are an important new class of
tau fibrillization inducer. Although their mechanism of
induction closely resembles that of AA, their adsorption
properties may differ substantially. To clarify this issue,adsorption of 124 pM anionic microspheres (90 nm di-
ameter; 12�A2/eq molecular area) onto grids was deter-
mined after 24-h incubation in the presence or absence
of 4 lM htau40C291A;C322A. In the absence of tau protein,
plots of interfacial microsphere concentration (CM) were
linear with dilution (Fig. 7A). This pattern, termed type
Fig. 7. Anionic microsphere adsorption isotherms. Anionic micro-
spheres (124 pM) were incubated (24 h at 37 �C) with (j) or without
(�) 4 lM htau40C291A;C322A under fibrillization conditions and then
treated with 2% glutaraldehyde, serially diluted in Dilution Buffer, and
adsorbed onto grids for 1min. The interfacial concentration of micr-
ospheres (CM; j;�) and tau filaments (Cf ; s) were then estimated
from electron micrographs. (A) Each point represents C measured as a
function of dilution (where a dilution of unity¼ 124pM microspheres).
Although microsphere adsorption was linear with dilution in the ab-
sence of tau (�; line represents linear regression), it was hyperbolic in
the presence of tau (j; line represents best fit to the Toth isotherm),
suggesting that microsphere adsorption was protein concentration
dependent. Under these conditions, Cf also varied hyperbolically with
bulk tau concentration (s; line represents best fit to the Toth iso-
therm). (B) Replot of CM versus Cf determined at different microsphere
concentrations (error bars were omitted for clarity). Because filaments
remained associated with microspheres after nucleation, Cf varied
linearly with CM.
C1 in the nomenclature of Giles, is typical of adsorbantswhen present well below saturation [41]. In contrast,
plots of interfacial microsphere concentration against
dilution were hyperbolic in the presence of tau (Fig. 7A).
Fitting the data to the Toth isotherm yielded a Toth
coefficient of 1.3� 0.4 and a saturating interfacial con-
centration of 1.28� 0.12microspheres/lm2. Toth coef-
ficients of �1 reduce to the Langmuir isotherm [32], as
reflected in the classic rectangular hyperbolic shape ofthe curve (Giles type L2). These data show that tau
protein influences microsphere adsorption behavior and
that assay of tau fibrillization using microspheres in-
stead of AA as inducer does not alleviate the nonlin-
earity produced by the different concentrations of tau
protein. Nonetheless, because filaments grow from in-
ducer surfaces and remain associated after nucleation
[9], microspheres could potentially serve as easily mea-sured internal standards for adsorption. To test this
hypothesis, the study was extended to include mea-
surement of interfacial filament concentration (Cf ). Re-
sults showed that Cf paralleled CM as a function of
microsphere dilution (Fig. 7A) and that plots of Cf
versus CM were linear at all dilutions examined
(Fig. 7B). These data confirmed that the Cf=CM ratio
was constant even when microsphere adsorption wassaturating and suggested that microspheres could be
used to normalize electron-microscopy-based length
measurements performed under nonlinear adsorption
conditions.
Discussion
Electron microscopy-based assays for tau fibrilliza-
tion differ from solution-based methods by employing
an adsorption step onto hydrophobic grids prior to
quantitation of reaction products. The data presented
here suggest that filament adsorption is linearly related
to concentration in solution up to saturation when
measured at constant pH, ionic strength, adsorption
time, and AA inducer and tau concentrations. This be-havior is maintained in the presence of varying con-
centrations of AA inducer, suggesting that filament
lengths can be assayed by electron microscopy when this
parameter is varied. Moreover, filament adsorption is
not modulated by Congo red-related dyes such as N744
up to 50 lM concentrations.
The major variable in assay performance appears to
be bulk tau concentration, which competes with fila-mentous tau adsorption on grids. Therefore, we propose
that tau adsorption phenomena reduce to a binary sys-
tem consisting of monomeric and fibrillized tau popu-
lations when examined at constant pH, ionic strength,
and adsorption time. The ratio of interfacial concen-
trations for two competing adsorbates (termed 1 and 2)
has been modeled by the expression [15]
� � � �244 M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246
C2
C01
¼ 1þ kd1k01
ka2ka1
C2
C1
þ 1þ ka2ka1
C2
C1
kexk01
C2; ð1Þ
where the ratio of interfacial concentrations (C) is a
function of each adsorbate�s bulk concentration (C) and
relative rates of absorption (ka), desorption (kd), ex-
change (kex), and transformation to an irreversibly ad-sorbed state (k01 in the case of the first adsorbate). The
absence of Vroman effects accompanying tau filament
adsorption suggests that exchange reactions for fila-
mentous tau are minimal over the 15-min time courses
investigated here (i.e., kex � 1). Furthermore, adsorp-
tion of filaments appears to be tenacious, suggesting that
rates of desorption also are minimal (i.e., kd � 1). If the
exchange and desorption rates of monomeric tau alsoare low, then the ratio of interfacial concentrations re-
duces to the ratio of bulk concentrations of each com-
ponent [15,40]. In other words, adsorption isotherms for
tau filaments reflect not only filament bulk concentra-
tion in solution but also the bulk concentration of
nonfibrillar tau. As a result, estimates of Cf differ when
constant amounts of tau filaments are adsorbed in the
presence of varying concentrations of total tau protein.These considerations extend to filaments of different
lengths also. Because the adsorption of higher-molecu-
lar-weight polymers is thermodynamically preferred
over the lower-molecular-weight fibrils, they would be
expected to exchange with shorter polymers over time
[42]. But kinetic curves show no signs of such behavior
(displacement), again presumably due to minimal de-
sorption and exchange reactions [43,44]. In any event,length distributions do not vary as a function of ad-
sorption time or concentration, suggesting that such
measurements are reliable when performed at constant
total tau concentration.
Published data are consistent with this model. First,
authentic filaments diluted in the absence of competing
substances have been shown to adsorb linearly with fil-
ament concentration [12]. This isotherm, termed C1 inthe Giles system [33], is typically observed at sorbate
concentrations far below saturation [41]. Thus, linearity
depends on the initial concentration of purified tau fil-
aments in the preparation. Second, filaments prepared in
vitro from synthetic inducers such as fatty acids adsorb
linearly as part of a multicomponent system so long as
the tau concentration is held constant. Thus measure-
ments of inducer efficacy at constant tau protein, forexample, are directly comparable [9]. Third, synthetic
tau filament adsorption is nonlinear when performed at
widely different total tau concentrations, which have
been used in studies characterizing tau fibrillization in
the presence of heparin [14]. Moreover, heparin-induced
reactions are typically conducted at 10–100 lM total tau
protein, far above the concentrations used here [6,14].
Although Vroman effects are not apparent up to 10 lMtau (the highest concentration used in our experiments),
use of higher concentrations may drive exchange reac-tions and further complicate quantitation of filament
lengths.
The nature of fibril–surface interaction may explain
the lack of the Vroman effect at low (610 lM) tau
concentrations. Although driving forces for adsorption
of proteins include hydrophobic and electrostatic inter-
actions [45], binding to the hydrophobic surface of
formvar/carbon-coated grids is presumably dominatedby hydrophobic interactions. Strong hydrophobic in-
teractions between the fibrils and the surface probably
contribute to tenacious fibril adsorption and the lack of
fibril displacement [29], which may further increase due
to glutaraldehyde treatment. Indeed, the Vroman effect
diminishes with increasing surface hydrophobicity
[15,29,46]. Tenacious adsorption to surfaces also is
promoted by entropy gains resulting from protein con-formational changes subsequent to adsorption [25].
However, changes in monomeric tau or tau filament
conformation on the grid surface are unlikely under
conditions used herein because samples were fixed with
glutaraldehyde prior to adsorption and because mono-
meric tau is natively unfolded [47].
Although the above considerations adequately de-
scribe the macroscopic behavior of tau adsorption,events at the molecular level are unclear. Indeed, the
molecular basis for continuous-shape adsorption iso-
therms is not understood for purified analytes let alone
the complex mixture described here [41]. But the Giles
type C2 isotherm and high Toth coefficient accompa-
nying dilution of tau fibrillization products are consis-
tent with secondary events accompanying adsorption so
that the effective surface area appears to increase [32,41].This behavior may be unique to surfactants such as AA
because anionic microspheres, a chemically distinct class
of fibrillization inducer, do not behave in this way, in-
stead adopting the classic Langmuir adsorption iso-
therm when diluted in the presence of tau protein.
In summary, accurate quantitation of filament length
distributions and total filament length can be performed
under conditions of constant bulk tau concentration.Interference from anionic surfactant (6200 lM) and
fluorescent dyes (650 lM) are minimal under these
conditions. Filament lengths can also be determined at
different total tau concentrations, but samples must be
diluted so that the interfacial tau filament concentration
is not saturating. Even then, care must be taken so that
competition from nonfibrillar tau is minimal. We pro-
pose two solutions to this problem. First, samples can bediluted into solutions containing glutaraldehyde-treated
tau monomer prior to adsorption to normalize for bulk
tau concentration. Filament lengths are linear under
these conditions, at least up to 10 lM bulk tau. Second,
tau can be fibrillized using anionic microspheres (which
are readily visualized in the electron microscope) instead
of fatty acids. Filaments grow from microsphere
M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246 245
surfaces and remain associated with them at equilib-rium. Thus interfacial microsphere concentration pro-
vides an internal standard for adsorption reactions in
the presence of competitive adsorbates such as mono-
meric tau protein. As a bonus, this strategy can be used
to normalize interfacial concentrations to the entire re-
action volume and therefore calibrate fibrillar tau with
regard to molar concentration.
Acknowledgments
This work was supported by Grant AG14452 (J.K.)from the National Institutes of Health.
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