2.1.1. Absorbance Measurements

To monitor the effect of pH on the tertiary structure of edG, we measured the absorbance spectra at a wide range of pH. The side chain of aromatic amino acids of protein acts as chromophore as it contains the conjugated double bond system [33]. The maximum absorption (λmax) in the UV region of the protein is extremely sensitive due to the change in the local milieu of the aromatic amino acid as it increases the solvent exposure that leads to a blue shift in the λmax [34]. The edG has five tyrosine and one tryptophan residues that give rise to maximum absorption (λmax) at 278 nm wavelength. The change in the molar absorbance coefficient of native and denatured protein at 278 nm (∆ε278) plotted as a function of pH, which probes the environmental changes of the aromatic group of the proteins and hence the native biological structure of the protein [35].

We did not find any significant change in the spectra of edG on increasing the pH from pH 2.0–3.0 and 8.0–12.0, which indicates intact native structure at these pH ranges (Figure 1). The protein does not show any scattering signals during the pH-based studies except at pH 4.0–6.0, indicating good solubility of the protein. However, the acid-induced loss of tertiary interactions occurs due to aggregation at pH values 4.0–6.0 that shows the complete distortion of spectra with a noticeable scattering signal in the range of 320–340 nm. Our finding from the spectral study confers that the edG is stable at physiological pH and highly acidic and basic pH values, but the loss in structure was observed at mildly acidic pH (4.0–6.0). From our study, we also conclude that at highly acidic and basic pH values, the edG does not show any type of aggregation; however, at mild acidic pH, a visible aggregation of protein was observed. The plot of ∆ε<sup>278</sup> versus pH shows no remarkable change in molar absorption coefficient in highly acidic and alkaline pH values (see inset of Figure 1). Hence, the loss of edG tertiary interaction at mildly acidic pH may be correlated with its biological activity, as suggested in our earlier publication [30].
