Self-Assembly of Synthetic and Biological Polymeric Systems of ...

Additional Supra-Self-Assembly of HSA J. Phys. Chem. B, Vol. 113, No. 36, 2009 12393
instrument (PSIA Inc., Seoul, Korea) equipped with a rigid
silicon cantilever of nominal spring constant of about 40 mN/m
and resonant frequency of 325 kHz. Immediately after incubation,
protein samples were diluted 20-400 times onto freshly
cleaved muscovite mica (from Sigma Chemical Co.) attached
to a magnetic steel disk which served as a sample holder. The
abrupt dilution of the samples immediately quenches the
concentration-dependent aggregation process. The AFM samples
were dried in air or under nitrogen flow when required. Control
samples (freshly cleaved mica, mica and buffer solution) were
also investigated with AFM to exclude possible artifacts.
Topography and phase-shift data were collected in the trace and
retrace direction of the raster, respectively. The offset point was
adapted accordingly to the roughness of the sample. The scan
rate was tuned proportionally to the area scanned and kept within
0.35-2 Hz range.
Rheometry. The rheological properties of the samples were
determined using a Bohlin CS10 rheometer with water bath
temperature control. Couette geometry (bob, 24.5 mm in
diameter, 27 mm in height; cup, 26.5 mm in diameter, 29 mm
in height) was used, with a 2.5 cm 3 sample being added to the
cup in the mobile state. Samples of very high modulus were
investigated using cone-and-plate geometry (diameter 40 mm,
angle 4°). A solvent trap maintained a water-saturated atmosphere
around the cells. Frequency scans of storage (G′) and
loss (G′′) moduli were recorded for selected protein concentrations
and temperatures with the instrument in the oscillatory
shear mode and with the strain amplitude (A) maintained at a
low value (A < 0.5%) by means of the autostress facility of the
Bohlin software. This ensured that measurements of G′ and G′′
were in the linear viscoelastic region. Measurements on solutions
of low modulus (G′ ) 1-10 Pa) which fell outside the range
for satisfactory autostress feedback were rejected.
Results and Discussion
Human serum albumin consists of 585 amino acids in a single
polypeptide chain, with a globular structure composed of three
main domains that are loosely joined together through physical
forces and six subdomains that are wrapped by disulfide bonds.
The protein contains 17 disulfide bridges and one free SH group.
Most of the HSA sequence (>60%) is arranged in R-helix
structure, with the subsequent tightening of its structure through
intramolecular interactions such as hydrogen bonds. Nevertheless,
amyloid-like formation of HSA has been reported under
different solution conditions. 41,42 HSA fibril formation usually
takes place in the absence of a lag phase, and the resulting fibrils
share the common structural features of “true” amyloid fibrils,
although their final morphology depends on the incubation
conditions. 42
Spherulite Formation and Characterization. The propensity
for spherulite formation was investigated by using optical
and confocal microscopy at pH 2.5, 5.5, and 7.4 at 65 °C upon
incubation in the presence of NaCl in the range 0-250 mM.
We have observed that spherulitic structures are found under
conditions where HSA fibrils were previously detected, 42 that
is, at physiological pH and under acidic conditions in the
presence of the highest added salt concentrations (100-250 mM
NaCl). Although data were not collected quantitatively, the
highest density of spherulites was observed at pH 7.4 in the
presence of 50 mM NaCl, and at acidic pH in the presence of
250 mM. This agrees with the maximum efficiency of fibril
formation previously reported. 42 In this regard, it has been
previously found41 that at both physiological pH and temperature
fibril formation is disfavored at high ionic strengths due to an
excesive shielding of repulsive electrostatic forces, which
involves an increase in rapid random protein aggregation. In
contrast, under acidic conditions fibril formation is continuosly
enhanced as the added salt concentration increases. This
difference has been assigned to the different nature and structure
of the protein precursor states, which leads to different fibrillation
rates, fibril conversion extents, fibrillation pathways, and
structures of resulting fibers. 44-46 In addition, no differences were
observed between the spherulites obtained in the various samples
studied regardless of the incubation conditions.
Analysis of the solutions at both pH 2.5 and 7.4 by polarized
optical microscopy allowed us to observe the presence of
classical Maltese cross extinction patterns (Figure 1a) as a key
mark of the presence of spherulites. This type of pattern was
not observed at pH 5.5. The observed spherulites possess sizes
ranging from 5 to 50 µm. In addition, the test tubes when placed
between crossed polarizers showed birefrigence from spherulites
in the samples (not shown). Spherulitic structures are detected
both in solution prior to gelification and embedded in an
isotropic gel matrix after gelification. It should also be noted
that nonspherulitic areas of the sample, which may contain fibrils
not assembled into spherulites, are not visibly birefringent,
indicating that polypeptide chains in this region are unoriented.
Similar spherulitic occurrences in protein gels were also found,
for example, for whey proteins, 47 -lactoglobulin, 29,30 and
insulin. 32,33
To confirm that the spherulites contain amyloid fibrils, they
were imaged by confocal microscopy using ThT as the
fluorescent dye, whose fluorescence is known to increase
markedly upong binding to amyloid fibrils. 48 In confocal mode,
the fluorescence emission was observed to come from the
spherulites (Figure 1b), which confirms they are composed of
amyloid fibers. 49 Confocal microscopy in transmission mode
resulted in an image similar to that obtained with an optical
microscope (Figure 1c), clearly displaying the presence of
spherulites as Maltese cross patterns of comparable sizes to those
observed by optical miscroscopy. The nonordered and nonfluorescent
core is probably due to nonpenetration of ThT. As this
core is present even after extensive incubation in the presence
of the dye, a more feasible explanation is the absence of
significant amounts of amyloid material in this core, that is, it
is formed before spherulitic growth takes place due to an
interplay between amyloid fibrillation and protein random
aggregation. 16 Aliquots of spherulitic solutions were also added
to solutions containing ThT. The enhanced fluorescence relative
to solutions of ThT and ThT containing freshly dissolved HSA
additionally proved the presence of amyloid structures forming
the spherulites (see Figure 1d).
To reveal the orientation of the fibrils within the spherulites,
insertion of a waveplate at 45° angle to the cross polars provided
confirmation of this radial arrangement (see inset in Figure 1a).
The four quadrants in the spherulites are now colored. The
appearance of the blue color indicates that the fast optical axis
of the spherulites is aligned with the fast optical axis of the
wave retardation plate. 34 The slow direction within the spherulite
is the radial direction and, therefore, indicates that the fibrils
lie radially within the spherulites, as seen previously for other
proteins such as -lactoglobulin, 20,31,32 R-L-iduronidase, 33
lysozyme, 18 and insulin. 16,34 Figure 1e shows that out from the
core the radially oriented internal structure appears to be strands
of fibril bundles (because individual fibrils are too narrow to
be seen optically) with slight curvature to their orientation and
without obvious branching. This is particularly viewable at the
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