Structurally modified lysozyme via solvent browning: Toward functional protein-based antioxidants

Hen egg white lysozyme is a well-characterized protein extensively studied for its structural and functional properties. Chemical modification strategies, including Maillard reaction, have been shown to alter lysozyme's structure and bioactivity. We previously identified a novel browning reaction initiated by simply dissolving proteins in a solvent mixture of dimethyl sulfoxide and acetone, resulting in time-dependent chromophore formation. Here, we applied this solvent-mediated browning to lysozyme and investigated the resulting structural and functional transformations. UV-vis spectroscopy revealed progressive absorbance increases and color intensification, plateauing after 48 h. SDS-PAGE analysis indicated partial dimerization, likely associated with disulfide bond disruption, as supported by thiol quantification. FTIR and CD spectroscopy showed loss of α-helical content, rearrangement of β-sheet structures, and overall loosening of the protein's secondary and tertiary architecture. Fluorescence quenching and reduced surface hydrophobicity were observed and found to be positively correlated. SEM imaging revealed morphological changes consistent with increased fibrillar content in browned samples. Functionally, browned lysozyme exhibited markedly enhanced antioxidant activity while displaying reduced antibacterial efficacy, though cytotoxicity remained negligible. These findings demonstrate that solvent-mediated browning represents a simple yet effective strategy for structurally reprogramming lysozyme and potentially other proteins, thereby expanding their functional versatility for food, biomedical, and pharmaceutical applications.