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.