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

Additional Supra-Self-Assembly of HSA J. Phys. Chem. B, Vol. 113, No. 36, 2009 12397
with the presence of relatively long, large, and thick aggregates
which seem to be composed, at least partially, of globular
aggregates (Figure 5b and c). Additionally, the size of the
elementary particles appeared to be larger than those formed at
lower added salt concentrations. A similar picture was observed
for the gel formed at acidic pH (not shown). Therefore, a
transition from fine-stranded to coarse gels with increasing ionic
strength seems to be caused mainly by kinetic effects without
accompanying fundamental changes in aggregation mechanisms.
58 After extensive dilution and sonication, rupture of these
types of aggregates in a variety of fibrillar structures rather than
in smaller spherical aggregates was observed by TEM and AFM
(see Figure 5d and e), as seen in previous studies. 59 The length
of the fibrils varies between 0.1 and 1-2 µm with a thickness
of 15-30 nm.
In contrast, at pH 5.5 a network of quasipherical protein
aggregates surrounded by liquid is formed; i.e., we observed a
particulate gel (see Figure 5f-h). The particle size varies
between ∼300 and ∼500 nm, which increases as the ionic
strength increases. This leads to fused long and thick aggregates
possibily composed of several particulates. In this regard, it has
been speculated that intermolecular disulfide bonding is involved
in connecting protein molecules within the particulates and that
the connections among them is a nonspecific physical crosslinking
without any specific connective sites. 58,60 ESEM also
indicates that an amount of residual monomeric protein was
left in the solutions (Figure 5g) and deposited between the
particulates. This gives the appearance of connected particulates
and, sometimes, apparently amorphous films. As commented
above, shifting the pH toward the isoelectric point decreases
the intermolecular electrostatic repulsion. This implies that
aggregation becomes faster, preventing the formation of highly
ordered nanostructures such as amyloid-like fibrils, but globular
shapes are formed.
Thus, it seems that the gelation of HSA is also pH-dependent
and the structures which form the gel depend on the solution
conditions. In this regard, in a recent report the capability of
several proteins such as -lactoglobulin, BSA, insulin, and
lysozyme among others to form either fibrillar or particulate
gels under partially denaturing conditions but at different pHs
has been confirmed. 61 This suggests that the formation of
particulates can be a generic property of all polypeptide chains
like amyloid fibrillation is. In this way, the results reported in
this work support this view.
Finally, in order to investigate the internal structure of the
fibrillar and particulate gels, attenuated total reflectance Fourier
transform infrared spectroscopy (ATR-FTIR) measurements
were made. All spectra were well resolved with clearly
distinguishable secondary structure signatures. In this way,
before incubation two major bands peaks in the second
derivative IR spectra in the spectral region of interest were
observed at pH 7.4: the amide I band at 1652 cm -1 and the
amide II band at 1544 cm -1 . This indicates the predominant
structural contribution of major R-helix and minor random coil
structures. 62,63 For the amide I band (see Figure 6a), a shoulder
at ca. 1630 cm -1 can also be observed in the second derivative
spectra, which is related to a low intramolecular -sheet content.
Additional peaks at ca. 1689 and 1514 cm -1 would correspond
to -turn and tyrosine absorption, respectively. 63 As the pH is
lowered, the remaining presence of the amide I and II bands
confirms that there is still a significant amount of R-helices,
even at the most acidic pH (see Figure 6a).
Following incubation at 65 °C and formation of the gel at
pH 7.4, a red-shift of the amide I band from 1652 to 1658 cm -1
Figure 6. Second derivative of FTIR spectra of HSA samples formed
at (a) pH 2.5, (b) pH 5.5, and (c) pH 7.4 in the presence of 50 mM
NaCl (s) before and ( ···) after incubation and gelation at 65 °C.
(1650 to 1656 cm -1 at acidic pH) and a blue-shift of the amide
II band to 1542 cm -1 (1540 cm -1 for pH 2.5) is indicative of a
certain increase of disordered structure (see Figure 6b). The
appearance of a well-defined peak around 1625 cm -1 (1624
cm -1 for pH 2.5) points to a structural transformation from an
intramolecular hydrogen-bonded -sheet to an intermolecular
hydrogen-bonded--sheet structure, 64 which is a structural
characteristic of the amyloid fibrils. The spectrum also shows
a high frequency component (∼1693 cm -1 ) that would suggest
the presence of an antiparallel -sheet. 65 In addition, a small
shoulder around 1534 cm -1 was also assigned to a -sheet. 66
In the case of particulate gels formed under incubation at 65
°C at pH 5.5, we could also observe a decrease and shift to
1657 cm -1 of the amide I band and an increase in the content
of -sheet conformation. This is indicated by the enhancement
of the intensity and further shift of the band positioned around
1626 cm -1 , in agreement with previous reports (see Figure
6c). 61,67,68 In addition, the proportion of -sheet content is lower
than that at pH 7.4 and 2.5. This corroborates that the
aggregation near the protein isoelectric point takes place faster
and nonspecifically which decreases the likehood of substantial
structural rearrangements during the aggregation process. In
contrast, amyloid fibrils and fibrillar gels resulted from partially
highly charged unfolded states, 41,42 which involve long-range
repulsion and slow aggregation occurring only when substantial
structural reorganization allows the formation of a favorable
structure, the cross- structure. In fact, when excess electrolyte
is added, aggregation becomes faster and the proportion of
-sheet structure decreases due to an enhancement of the
aggregation rates. 46
Conclusions
In this work, we have described the existence of suprafibrillar
assemblies formed by the protein human serum
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