Biophysical Assessment of Protein Stability in Ethanol-Stressed Environments via UV Absorption and Fluorescence Spectroscopies

Abstract

Maintaining the structure and functionality of proteins is crucial in applications ranging from food preservation to pharmaceutical formulation. Ethanol, while commonly used as a solvent and preservative, can induce structural changes in proteins depending on its concentration and the specific structure of the protein itself. This study investigates the structural effects of ethanol on three types of model proteins, namely bovine serum albumin (BSA), beta-Lactoglobulin (beta-Lg), and beta-Casein (beta-Cn), by using UV-Vis spectroscopy and fluorescence spectroscopy. The conformational responses of proteins in water-EtOH solutions of various ethanol concentrations (0-10 %, v/v) were analyzed through absorbance and emission spectral changes. At increasing ethanol concentration, UV absorption data showed distinct protein-dependent spectral changes. beta-Lg and beta-Cn exhibited strong hypochromism (an absorbance decrease of similar to 25 %) and red-shifting at 222 nm and 220 nm, respectively, indicating partial unfolding and solvent exposure of aromatic residues. BSA demonstrated subtle changes, and consistent quenching in fluorescence with a continuous blue-shifting to 330 nm, suggesting a moderate overall stability and local rearrangements in its structure. beta-Cn exhibited red-shifted fluorescence and quenching, reflecting its flexible, disordered structure and heterogeneous response to solvent conditions. Statistical analysis revealed that while fluorescence spectroscopy was highly sensitive to the intrinsic differences between proteins (p < 0.001), the ethanol-induced conformational changes were too subtle to be detected as a statistically significant treatment effect. The consistency of these trends indicates a rational underlying mechanism of interaction. This reflects the subtle nature of the effect at the tested concentrations rather than the absence of an effect. Moreover, these results unveil the protein-specific effects of ethanol and strongly emphasize the importance of solvent composition in maintaining protein integrity. Ethanol concentrations up to 5 % may offer protein stability whereas high ethanol levels (>= 5-10 %) promote structural perturbations. These results will be useful for both basic scientific research, such as biophysical studies and the advancement of optical techniques, and various industrial uses.