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

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below, the first increase is related to the presence of progressively
interconnected aggregates from the reduction in solvent polarity,
which originates subtle changes in the protein secondary structure
composition favoring the entanglement of protein aggregates. The
subsequent intensity decrease at larger alcohol concentrations (>
50% v/v) originates from the formation of well-dispersed
aggregates (short fibrils, see TEM pictures below) as a
consequence of additional changes in protein secondary structure
(as confirmed CD data below). The final scattered intensity
increase at the largest ethanol contents arises from the presence of
more numerous and longer aggregated structures in solution.
These observations are in contrast with the behavior observed
at 25 ºC (see Figure 1d). For samples containing low ethanol
contents, their low scattering intensity suggests that almost no
aggregation occurs, whereas at higher ethanol contents (>40%
v/v) the rise in the scattered intensity profile corroborates the
presence of aggregates as a consequence of the screening of
electrostatic repulsions between protein molecules as the solvent
permittivity decreases. Additional confirmation is given by CD
and ThT fluorescence data below.
On the other hand, comparison of scattered intensities at
acidic and physiological pH shows the scattered intensity at the
former pH is, in general, lower than that at the latter at the same
mixed solvent conditions (see Figure 1). This is a consequence of
the lowering of intermolecular interactions due to protonation of
positively charged residues. 57 Also, when comparing the effect of
temperature on the scattered intensity profiles, we can observe
that incubation at 25 ºC involves a lower scattered intensity at
both pH in the whole range of solvent compositions. It is known
that hydrogen bonding is weakened as temperature rises, but
hydrophobic interactions become strengthened. 58 As the heating
process proceeds, destabilization of the protein helical structure
of HSA occurs though hydrogen bonding weakening, as denoted
by the loss of the minima at 208 and 222 nm typically assigned to
α-helices in the HSA native state in the CD profiles of HSA
samples at high temperature (see Figure 3); then, previously
buried aminoacid residues can be now exposed to solvent as
follows from changes in protein conformation from CD data (see
below), which results more prone to aggregation as observed
from the SLS data (and further confirmed by TEM pictures
below).
Aggregation kinetics.
Data analysis of scattered intensity plots with a sigmoidal-like
profile is consistent (also according to spectroscopy and TEM
data), at a first approximation, with the following kinetic scheme:
M ↔ I ↔ nucleus → fibrils
where M is the monomer and I is the intermediate. Thus, the
scattered intensity as a function of time was fitted to the
following equation:
(2)
where Y is the scattered intensity, x the time, and xo is the time to
reach 50% of maximal scattered intensity. Thus, the apparent rate
constant, kapp, of fibril growth is given by 1/τ, and the lag time by
xo - 2τ. 59 Lag times and apparent first-order rate constants were
determined from curve fits and shown in Table S1 in ESI. In
general, an increase in the alcohol concentration resulted in
longer lag times and larger apparent rate constants. Also, the
aggregation kinetics at physiological pH appears to be faster than
under acidic conditions. This behavior is probably related to the
existence of electrostatic repulsions between protein molecules
and to an enhanced solubility of the initial protein clusters under
acidic conditions. Finally, it is necessary to mention that two
growth rates were obtained at high ethanol concentrations upon
incubation at acidic conditions and 25 ºC. This fact is originated
by the presence of intermediate oligomeric structures along the
HSA aggregation pathway under these solution conditions, as
discussed elsewhere. 40
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Aggregate size distribution.
Figure 2 shows some examples of population size
distributions for samples incubated under the different solution
conditions. At 65 ºC and physiological pH, two different
populations are well differentiated and present under all mixed
solvent compositions (Figure 2a): A peak at large sizes (ca. 400-
1000 nm) assigned to protein aggregates, and other at smaller
sizes (at ca. 20 to 40 nm) probably representing protein
clusters/oligomers. Also, it is observed that a third population of
aggregates with very large sizes (∼ 20 µm) appears at an ethanol
concentration of 80% (v/v). This feature confirms that supraaggregation
of protein aggregates is favored at high alcohol
contents under the present conditions, in agreement with SLS
data, and as further confirmed below by TEM pictures. For
samples incubated at 25 ºC and physiological pH and at 65 ºC and
pH 2.0 (Figure 2b-c), a progressive increase in the size of protein
aggregates takes place as the ethanol concentration increases, as
denoted by the existence of bimodal distributions. Only at
intermediate ethanol concentrations the referred size increase
involves the superimposition of population sizes corresponding to
large protein aggregates and oligomeric structures, creating a
wide population distribution (but still bimodal, as observed after
deconvolution). In contrast, incubation at pH 2.0 and 25 ºC
results in the simultaneous decrease of the peak height
corresponding to the smallest sizes and the increase of that
corresponding to the biggest ones. This fact indicates a
progressive evolvement of protein monomers and small
oligomeric structures to aggregates of larger sizes as the ethanol
concentration in the mixed solvent increases (Figure 2d),
corroborating SLS data.
Regarding the hydrodynamic radii derived from the size
distributions depicted in Figure 2a-d, they can be grouped into
three average values corresponding to rh,app of ca. 400-1000 nm,
20-50 nm, and 2-5 nm. At this point, it is necessary to remind that
DLS sizes are obtained assuming a spherical shape, so these
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