Effect of shear on the aggregation of BSA at different
temperatures
After conducting the thermal stability study, we chose three temperature
below 65°C (55, 60 and 65°C) to investigate the thermomechanical effects
at a constant shear rate of 300 s-1. Figure 3 shows
Th-T flourescence intensity for the samples collected at different time
intervals during the shearing. The aggregation was obsrved for the
sheared smaples at 60°C and 65°C; although the Th-T intensity and
aggregation rate were enhanced at the higher temperature. At these
conditions, the BSA solution got exposed to enough energy from both
shear and thermal processes, which led to the disruption of
intramolecular bonds and exposure of the hydrophobic core. This
ultimately resulted intermolecular interactions leading to the
aggregation of the protein (Militello et
al. 2003). While at 55°C, the Th-T intensities were much lower as
compared to the other two temperatures, which indicated the absence of
aggregates. The hydrodynamic diameter, dH , values
were also correlated with the Th-T fluorescence data (Figure 3 (a)). At
55°C, BSA was present in monomer or dimer forms; howeverdH values were enhanced about 4 and 7 folds when
sheared at 60°C and 65°C, respectively. These observations reflect the
vulnerability of a protein towards aggregation after exposing to shear
even below its melting range (63 °C).
Further, rate of aggregation (\(k\)) was deciphered by fitting the Th-T
fluorescence intensity (\(I_{\text{t\ }}\)) with a single exponential
expression as \(I_{\text{t\ }}=I_{\max}(1-e^{-kt})\)(Holm et al. 2007;
Singh et al. 2017;
Solá et al. 2006). The experimental data
fitted well to this 1st order kinetic model withR 2 in the range of 0.90 to 0.99. Fitted data
are shown as dotted lines in Figure 3 (b). The rate constant \((k)\)values were found to be 0.076 ± 0.01 min-1, 0.024 ±
0.005 min-1 and 0.021 ± 0.005 min-1for the samples sheared at 55°C, 60°C and 65°C, respectively. The \(k\)values were compared with that in case on only thermal treatment at the
same temperature. The \(k\) values were 1.5 times enhanced for the
thermomechanical (sheared) samples as compared to thermal treated
samples at 55°C, 60°C and 65°C, respectively. These findings indicate
the presence of at least two steps during the aggregation process as\(N\left(\text{native}\right)\leftrightarrow U(unfolding)\leftrightarrow A(fibrillation)\)(Danielsson et al. 2015;
Haynes and Norde 1995). At the
temperature 55°C (below melting point), the protein was not completed
unfolded as only a slight change in dH was
observed. However, aggregation was observed when sheared at 60°C and
65°C, which followed 1st order kinetics (Figure 3
(b)). This agreed to the second step of the aggregation process.
Furthermore, the activation energy (Ea ) were
calculated from the slope of Arrhenius plot (lnk vs 1/T)
(Singh et al. 2017) and found to be ∼95
and ∼81 kJ/mol for the thermal and thermomechanical treatments,
respectively. The Gibbs free energy \(G\) was estimated as\(RT\text{lnk}\). \(G\) value was increased by 2.5 kJ/mol and 4.2 kJ/mol
at 65°C as compare to 55°C for the thermal and thermomechanical treated
samples, respectively. This agreed with the decreased stability at
higher temperature and under shearing. The magnitude of energy was found
to be comparable with the heat transferred during the thermal treatment
at 75°C (see the thermal energy section). This indicated that extra
energy during the shearing even at a lower temperature 65°C is
compensated from the dissipation energy, which is discussed in the next
section.