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

Fibrillation Process of Human Serum Albumin J. Phys. Chem. B, Vol. 113, No. 30, 2009 10523
Figure 1. Time evolution of ThT fluorescence in HSA solutions
incubated at 65 °C at (a) pH 7.4 and (b) pH 3.0 in the presence of (9)
0, (O) 20, (2) 50, (3) 100, ([) 150, and (+) 250 mM NaCl.
an extended less-ordered configuration (E-state) below pH 3.5
with lesser helical content. 37-39 On the other hand, 65 °C was
chosen as the incubation temperature, provided that HSA
temperature-induced denaturation takes place through a twostate
transition with a first melting temperature, Tm, of∼56 °C
and a second Tm of ∼62 °C 35,40,41 as a consequence of the
sequential unfolding of the different domains of the protein, in
particular IIA and IIIA subdomains: at 50 °C, a reversible
separation of site I and II occurs; below 70 °C, the irreversible
unfolding of site II is present, while increasing the temperature
over 70 °C or higher results in the irreversible unfolding
of site I. 42 Moreover, as the pH becomes more acidic, Tm
becomes lower. 35
Amyloid Formation Kinetics. When HSA solutions (20 mg/
mL in the presence of 0, 20, 50, 100, 150, or 250 mM NaCl)
were incubated at room temperature up to 300 h, the ThT
fluorescence emission intensity was negligible at pH 7.4 whereas
a slight increase was observed at pH 3.0 as a consequence of a
little gain in -sheet structure due to the formation of some
oligomeric aggregates (data not shown). This suggests that HSA
was not capable of forming amyloid fibrils or other types of
aggregates with significant -sheet structure under these conditions.
In contrast, a time-dependent increase in ThT fluorescence
is observed when the different HSA solutions were incubated
at 65 °C (see Figure 1), in accordance with fibril formation.
Congo Red (CR) absorption was also used to corroborate the
formation of these fibrils in HSA solutions, which display a
red-shift of the differential absorption maximum from 495 to
ca. 530 nm at 65 °C (see Figure S1 as an example).
In general, the kinetics of HSA fibrillation under the present
solution conditions at 65 °C involved the continuous rising of
TABLE 1: Kinetic Parameters of the Self-Assembly Process
of HSA Solutions
pH NaCl (mM) ∆F ksp (h-1 ) n
7.4 0 83 0.019 0.56
20 82 0.040 0.70
50 83 0.084 0.92
100 57 0.068 0.80
150 52 0.056 0.77
250 46 0.034 0.70
3.0 0 6/22 0.144/0.005 1.25/3.60
20 44 0.008 1.25
50 57 0.010 0.96
100 73 0.012 0.95
150 77 0.024 0.93
250 56 0.233 1.10
ThT fluorescence during the early periods of incubation until a
quasi-plateau region was attained in the time scale analyzed,
as occurred for different proteins such as BSA, 32 acylphosphatase,
7 or ovoalbumin, 33 and it exhibited no discernible lag
phase. This absence may be related to the fact that the initial
protein aggregation is a downhill process, which does not require
a highly organized and stable nucleus. This was confirmed by
the absence of any remarkable effect on the fluorescence curves
when protein seeds are added to protein solutions followed by
subsequent incubation (see Figure S2 in the Supporting Information).
It has been suggested that large multidomain proteins
such as BSA are able to form propagation-competent nucleuslike
structures (oligomeric structures), since there is no energy
barrier to impede aggregate growth. 32 As has been discussed in
detail previously before, 32,36 HSA forms these oligomers upon
very short incubation times, which usually occurs by a mechanism
of classical coagulation or downhill polymerization. 44
Nevertheless, a different behavior was found at acidic pH in
the absence of added electrolyte: After a small increase in ThT
fluorescence at very short incubation times, there exists an
almost plateau region (between 24 and 100 h approximately),
from which the ThT fluorescence starts to increase again (see
Figure 1b). This plateau region was identified as a lag phase,
as confirmed by a fibril seeding growth process (see Figure S3
in the Supporting Information). This might well indicate that
the formation of oligomeric structures might need more time
to be developed and/or persist for longer times due to their
enhanced solubility under acidic conditions in the absence of
added salt, so oligomers need to reach a certain number/size to
change the energy landscape of the system and promote further
aggregation.
Figure 1 also shows that amyloid formation was favored and
took place progressively quicker as the electrolyte concentration
increased from 0 to 50 mM NaCl at pH 7.4 and at the whole
concentration range studied at pH 3.0, as denoted by the
increment in ThT fluorescence intensity and the subsequent
presence of a plateau region at shorter incubation times. This
points out that the increasing hydrophobicity originated from
the screening of repulsive electrostatic interactions between
protein molecules is capable of stabilizing the -sheets. 36,45-48
In contrast, a progressive decrease in ThT fluorescence intensity
is observed at electrolyte concentrations larger than 50 mM at
physiological pH, with the plateau located at shorter incubation
times. This decrease can be related to the formation of bundles
of shorter fibrils and to the presence of amorphous aggregates
(with larger R-helix and unordered conformation contents, as
will be shown below), and is a consequence of an excessive
shielding of intermolecular electrostatic forces, which is accompanied
by an additional enhancement of the solution
167