Re-entrant condensation was observed upon mixing the tri-valent salts (YCl3 and LaCl3) with BSA (see the supplementary file) [42]. Upon mixing the salts with protein, a turbid-like solution was observed within a certain range of salt concentration, and below and above that salt concentration protein solution becomes transparent. For Y3+, the protein turbidity was observed around 3-5 mM salt concentration, whereas, for La3+, it was observed at around 1-3 mM salt concentration. The UV-vis absorption spectrum of BSA at room temperature (25ºC) is shown in Fig. S2. BSA shows absorption at around 278 nm [42]. Upon mixing the tri-valent salts (Y3+ and La3+), the absorption of BSA is not affected, but the absorption in the optical region, i.e., at around λ ≈ 630 nm, is affected. The variation of optical absorption is shown in Fig. 1. Fig. 1(a) and (b) corresponds to the variation of the optical absorption of BSA at a wavelength of λ ≈ 630 nm, and for the three different temperatures of 5, 15, and 25°C in the presence of 0-20 mM yttrium and lanthanum, respectively. From the figure, it is clear that in the re-entrant region, i.e., when protein solution becomes turbid, the optical absorbance becomes maximum. The rate of enhancement is also increased for lowering the solution temperature.
The fluorescence emission of BSA at room temperature (25ºC) is also shown in Fig. S2. BSA shows an emission at around 345 nm for an excitation at 278 nm. The variation of the maximum emission intensity, i.e., emission at 345 nm, at room temperature (25°C), in the presence of yttrium (Y3+) ions is shown in Fig. 2(a). The enhancement of emission intensity is observed in the re-entrant region. The emission intensity of the same samples was recorded for other temperatures also, and the variation of the maximum emission intensity is also plotted in the same figure.
Like yttrium, the fluorescence emission of BSA in the presence of La3+ was also observed. The maximum emission intensity of BSA in the presence of La3+, for different temperatures, is shown in Fig. 2(b). From both the figures, it can be observed that the emission intensity becomes maximum in the re-entrant phase region. Along with this, it is observed that the emission intensity is always higher for lower temperature. This can be further understood from Fig. 3. In this figure, the maximum emission intensity of BSA with temperature variation is plotted for a particular salt concentration. Fig. 3(a) and 3(b) corresponds to the variation of emission intensity of BSA for different temperatures, in the presence of Y3+ and La3+, respectively. Though the intensity variation is less but the decrement of the maximum emission intensity with increasing temperature is clearly visible.
The temperature dependence of the fluorescence emission can be utilized for the measurement of the activation energy (Ea) of the protein within the specific temperature range [43]. The activation energy is associated with the interconversion between the fluorescent and non-fluorescent state of the protein [44]. The activation energy can be calculated from the Arrhenius equation, which can be written as [44, 45]:
$$ln\left(\frac{{F}_{0}}{F}\right)= A-\frac{{E}_{a}}{RT} \dots \left(1\right)$$
where, F0 is maximum fluorescence intensity at lowest temperature and F is the maximum fluorescence intensity at other temperatures. A is exponential factor, R is the universal gas constant, and T is the absolute temperature. Using the equation, the variation of emission intensity with the reciprocal of temperature (1/T) is plotted in Fig. 4. The Arrhenius plot of BSA in the presence of various concentrations of yttrium and lanthanum ions are plotted in Fig. 4(a) and 4(b), respectively.
From the slope of the linear fitting of each plot, the activation energy corresponding to the particular samples are calculated and tabulated in Table 1. The variation of \({E}_{a}\) of the BSA protein in the presence of yttrium and lanthanum salts of different concentrations are plotted in Fig. 5(a) and 5(b) respectively.
Table 1
The values of activation energy calculated from Arrhenius plot for BSA in the presence of yttrium (Y3+) and lanthanum (La3+) ions.
Yttrium (Y3+) concentration (mM)
|
Activation energy (\({E}_{a}\)) with error (kJ mol−1)
|
Lanthanum (La3+) concentration (mM)
|
Activation energy (\({E}_{a}\)) with error (kJ mol−1)
|
0
|
3.48 ± 0.612
|
0
|
3.48 ± 0.612
|
1
|
4.17 ± 0.439
|
1
|
3.53 ± 0.210
|
3
|
5.15 ± 0.457
|
3
|
3.98 ± 0.251
|
5
|
5.14 ± 0.437
|
5
|
7.21 ± 0.774
|
7
|
3.56 ± 0.690
|
7
|
6.61 ± 1.503
|
10
|
3.87 ± 0.707
|
10
|
4.23 ± 0.785
|
20
|
1.75 ± 0.235
|
20
|
4.26 ± 0.220
|
From the analysis, it is thus clear that in the presence of both the salts, the activation energy of BSA protein becomes maximum in the re-entrant region, and takes the values of ≈ 5.1 and 7.2 kJ mol−1 for Y3+ and La3+ ions respectively. The activation energy associated with the fluorescence of pure BSA in the above-mentioned temperature range is obtained as ≈ 3.48 kJ mol−1. The activation energy as obtained is associated with the delicate micro-structural modifications around the tryptophan residues with the variation of surrounding physical or chemical environments. When the temperature of the protein solution is reduced, the fluidity of the solution also decreases [43], and due to the less mobility of the constituent molecules, the enhancement in the fluorescence emission is observed. On the other hand, due to the increment of temperature, the rotations and vibrations among the fluorophores may enhance and as a result the non-radiative decay increases and fluorescence intensity decreases [46]. In the presence of the trivalent ions, it was proposed that the apparent size of the constituent BSA solutes in the aqueous solution enhances and the mobility of the proteins decreases, and as a result the enhancement in the fluorescence emission is observed [42]. Thus, the effect of trivalent ions under re-entrant condition is nearly similar to the effect of reduction in temperature of the BSA solution. Thus, the activation energy as obtained at condensed phase is higher than the transparent phase of the protein solution. The occurrence of the lower mobility or higher viscosity of the solution leads to the increment of activation energy in the re-entrant region.