Figure 1. Temperature sweep test for determining the thermal stability of BSA solution at three different ranges of (a) 25-50-25°C, (b) 25-65-25°C, and (c) 25-75-25°C at the rate of 1°C/ min; (d) Th-T fluorescence intensity and the corresponding hydrodynamic radii,dH , of the samples subjected to the above temperature sweep tests.
Apart from the loss of reversibility, aggregation behavior of BSA was also monitored. Figure 1(d) shows the Th-T fluorescence data and corresponding dH values of the samples subjected to the above three temperature sweep tests. No increase in Th-T fluorescence intensities was observed for the 25-50-25 °C hysteresis loop. Similarity, the corresponding dH values remained constant (5.5±0.2 nm). This indicated the absence of any unfolding and aggregation of BSA upto 50 °C. However, at other two higher temperature ranges (25-65-25 °C and 25-75-25 °C), the fluorescence intensities were considerably enhanced, which confirmed the formation of aggregates (Pandey et al. 2012). Thioflavin T is a benzothiazole dye and binds to the amyloid fibrils (aggregates) with the cross β-sheet structures and generates an enhanced flourescence (Khurana et al. 2005; Wolfe et al. 2010). Congruently,dH values were also increases by two and three folds at 65 °C and 75 °C, respectively. These observation highlighted that the thermal treatment above the melting temperature (63 °C) (Pal et al. 2020) of BSA caused the unfolding and aggregation.
The thermal energy supplied during the heating process i.e. enthalpy (\(H\)) was calculated as \(C_{\text{pmixture}}T\). The heat capacity of BSA solution was calculated as\(C_{\text{pmixture}}=y_{\text{BSA}}C_{\text{pBSA}}+y_{\text{water}}C_{\text{pwater}}\)(Smith 1950). \(C_{\text{pwater}}\) and\(C_{\text{pBSA}}\) were taken as 4.18 J/g-K (75.24 J/mol-K) and 1.66 J/g-K (108.6 kJ/mol-K), respectively (Kong et al. 1982; Solá et al. 2006). Here we have assumed constant \(C_{p}\)values in this study, but it varies with temperature. \(H\) values for 25-50°C, 25-65°C and 25-75°C ranges were calculated as 1.9, 3.0, and 3.8 kJ/mol, respectively. For an open system, heat transfer, \(Q\), is equal to \(H\). Hydrogen bond energy for α-helix in water environment is reported to be 1.93 kJ/mol (Sheu et al. 2003). Hence, a higher energy input at 65 and 75 °C broke the intramolecular bonds, which resulted into unfolding and intermolecular interactions (aggregation).
To monitor the secondary structure of the samples, the far-UV CD spectra have been recorded from 260-190 nm. Figure 2 (a) shows the CD spectra of the native BSA along with the temperature ramped samples upto 50°C, 65°C and 75°C. CD spectra of native BSA consisted of two characteristics negative band of α-helix at around 208 nm and 222 nm and a positive band near about 195 nm (Chen et al. 2015; Paul et al. 2013). As the temperature ramp increased, a decreased in the ellipticity value was observed signifying the loss of the α-helix content. Also, the peak shifts towards 217 nm and 196 nm were observed (Jayamani and Shanmugam 2017), which signify the formation of a β-sheet structures (Kamada et al. 2017; Pandey et al. 2012). Also the MRE values at 222 nm are plotted as a function of temperatures (Figure 2 (b)). A substantial increase in MRE values for the samples treated at 65°C and 75°C. The folded fraction (\(\mathbf{\alpha}\)) for the samples is determined as\(\mathbf{\alpha=(}\mathbf{\theta}_{\mathbf{t}}\mathbf{-}\mathbf{\theta}_{\mathbf{u}}\mathbf{)/(}\mathbf{\theta}_{\mathbf{f}}\mathbf{-}\mathbf{\theta}_{\mathbf{u}}\mathbf{)\ }\)(Greenfield 2006). Hereθt is the MRE value at the desired state of the sample. θu and θf are the MRE values of the unfolded and folded states, respectively. The loss of helicity was found to be 30% and 44% at 65°C and 75°C, respectively. These data correlated with the enhanced Th-T fluorescence intensity and the corresponding changes in hydrodynamic diameter (Figure 1(d)).