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

10526 J. Phys. Chem. B, Vol. 113, No. 30, 2009 Juárez et al.
Figure 4. Second derivative of FT-IR spectra at 65 °C and (a) pH 7.4 and (b) pH 3.0 in the presence of (1) 0, (2) 20, (3) 50, (4) 100, (5) 150, and
(6) 250 mM NaCl after incubation.
Figure 5. Time evolution of Trp fluorescence of HSA solutions upon
incubation at (a) pH 7.4 and (b) pH 3.0 in the presence of (9) 0,(O)
20, (∆) 50, (1) 100, and (]) 150 mM, and (b) at pH 3.0 and 25 °C in
the presence of 50 mM NaCl.
contents, with the former mainly decreased to be converted into
the -sheet structure. Moreover, coil and turn conformations
also change throughout the incubation process, increasing
slightly their significance.
On the other hand, increases in the -sheet content become
more important as the electrolyte concentration increases, but
only up to 50 mM NaCl at physiological conditions and in the
whole concentration range at acidic pH, for which these
variations were lighter. In this regard, fully mature amyloid
fibrils could not be formed at low electrolyte concentrations at
acidic pH, provided that effective interactions between -strands
are not allowed due to electrostatic repulsions: recent studies
using hydrogen exchange and proteolysis have indicated a less-
ordered structure for protofilaments compared with mature
amyloid fibrils, 54,55 in agreement with their lower -sheet content
suggested by CD and ThT binding studies, as in the present
study. On the other hand, certain recovery of R-helical content
is observed at pH 7.4 at 150 and 250 mM NaCl concentrations,
which agrees with the decrease in ThT fluorescence intensity
observed at these concentrations, as commented previously.
FT-IR spectra recorded for HSA solutions under different
conditions at the beginning and the end of the incubation process
additionally corroborated ThT and CD data. Figure 4 shows
the second derivative of the HSA solutions at 65 °C atpH7.4
and pH 3.0 for different electrolyte concentrations. Following
incubation at 65 °C, a red-shift from 1652 to 1658 cm -1 (see
Figure S4 in the Supporting Information for the FT-IR spectrum
of native HSA) and a decrease in intensity of the amide I band
occur up to 50 mM NaCl at physiological conditions, which
are indicative of the presence of a certain increase of disordered
structure, as also seen by far-UV CD. For pH 3.0, this shift is
more progressive along the whole added salt concentration
range. The appearance of a well-defined peak around 1625 cm -1
points out a structural transformation to an intermolecular
hydrogen-bonded--sheet structure, 56 which is a structural
characteristic of the amyloid fibrils and is present at both pH
values. The spectra also show a high frequency component
(∼1693 cm -1 ) at these electrolyte concentrations that would
suggest the existence of a -sheet in an antiparallel configuration.
57 The decrease of the peak intensity at 1657 cm -1 and the
increase at 1625 cm -1 as the electrolyte concentration increases
from 0 to 50 mM NaCl at pH 7.4 support that the major
structural transition is from R-helix f -sheet and that
intermolecular interactions are favored at low and medium salt
concentrations, in agreement with CD data. In contrast, the
decrease in the band at 1625 cm -1 and the blue shift of the
band at 1542 cm -1 (not shown) at larger electrolyte concentrations
at this pH point out a lower -sheet configuration and a
further unordered conformation, which confirms that large
electrolyte concentrations do not facilitate amyloid formation,
in agreement with CD and ThT fluorescence data.
Tryptophanyl fluorescence data showed that when temperature
is raised to 65 °C, a reduction in fluorescence intensity and a
hypsochromic shift were observed, as noted in detail previously.
36 Nevertheless, we found that when incubation proceeds
at electrolyte concentrations larger than 100 mM NaCl at pH
7.4, a slightly lower decrease in fluorescence intensity takes
place, which agrees with the assumption that less ordered
amyloid-like fibrils are formed under these solution conditions
in coexistence with amorphous aggregates. In contrast, at acidic
pH, the decrease in fluorescence is also slightly larger as the
170