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

12394 J. Phys. Chem. B, Vol. 113, No. 36, 2009 Juárez et al.
Figure 1. (a) Optical microscopy images of grown spherulites of HSA at pH 7.4 in the absence of added NaCl at 65 °C after 72 h of incubation
under cross-polarizers. The inset indicates the radial arrangement of fibrils through insertion of a waveplate at 45° between the polarizers. (b)
Fluorescence image in the confocal mode of spherulites using ThT as a probe. (c) Confocal image in the transmission mode of spherulites between
cross-polarizers. (d) ThT fluorescence upon incubation in a solution without HSA, containing native HSA and containing spherulites. (e) Optical
microscopy image of HSA spherulites without polarizers. Scale bars are (a) 50, (b) 200, and (c and e) 100 µm.
outer edges of the spherulites, where the fibril bundles are
slightly separated from one another.
The spherical morphology of spherulites was also observed
by ESEM (see Figure 2), with sizes similar to those derived
from optical and confocal microscopy. Their surface appears
to have a texture on the length scale of several micrometers
but shows no obvious orientational order. Some cracks on the
particle surface appeared as a consequence of the dehydration
process in ESEM. Unfortunately, we could not observe if the
spherulites formed display a radial internal order, since internal
cracking of spherulitic structures was not achieved.
Direct measurement of spherulitic solutions was not posible
by TEM provided that a film was formed on the grids which
do not allow visualization. In this way, dilution and sonication
of the spherulitic solutions were performed in order to investigate
the samples by TEM. This treatment resulted in the
disruption of the spherulites and the appearance of amyloidlike
fibril structures, as seen in Figure 2d. This fact further
confirms that spherulites are mainly composed of amyloid fibrils.
Gel Formation and Characterization. Prolonged incubation
of solutions containing 0.3 mM HSA (or 0.8 mM where
corresponds) at either pH 7.4, 5.5 or 2.5 at 65 °C in the presence
of different amounts of added salt leads to the formation of
gels. In this work, we have only analyzed solution conditions
previously studied for amyloid fibril formation. 41,42 Significant
differences were observed in the association properties of the
different protein solutions. In this way, we have observed that
at pH 7.4 and 5.5 gels were formed at electrolyte concentrations
larger than 20 mM with gelation times decreasing from several
days to hours as the solution ionic strength increases (or the
protein concentration rises). On the other hand, under acidic
conditions gels were only formed at the highest electrolyte
concentration analyzed, 250 mM NaCl at 0.3 mM HSA. In
general, it seems that gel formation is favored by the screening
of electrostatic interactions between protein molecules, which
favors protein aggregation. 45,46 Nevertheless, protein interactions
at acidic pH are weaker than those at physiological pH due to
the initial different conformational state of the protein (Expanded-E
state under acidic conditions), which imparts a larger
solubility to the acid-denaturated protein molecules as a result
of electrostatic repulsion between them due to an increase of
surface-charged groups.
Figure 3a shows cure curves (G′, the storage modulus, versus
time) for gelling HSA solutions at pH 7.4, 5.5, and 2.5 in the
presence of 100 mM NaCl at 65 °C. To follow the protein
gelation in a suitable time scale for the experiment, we increased
the protein concentration up to 0.8 mM. The obtained cure data
are typical of a sol-gel transition of biopolymer solutions: The
elastic component of the shear modulus starts small but
undergoes a sudden increase at a critical time and, then,
ultimately almost levels off at longer times. For pH 7.4 and
2.5, the behavior is similar to that expected for a strongly gelling
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