Analyzing synergy in combined influence of sorbitol and glycine betaine on protein Stability: Thermodynamic and mechanistic insights

The cells of many organisms have mixtures of organic osmolytes, which raises the question of whether the action of the osmolytes on protein's stability is dependent or independent in nature. To resolve this query, it is essential to formulate a system that allows the investigation of the ability of osmolytes to fold or unfold a protein. Such stabilizing osmolytes include numerous amino acids and their derivatives, polyols, and methylamine molecules. Polyols and Glycine Betaine (GB) are the most efficient osmo protectants reported till date. The influence of sorbitol and GB on the stability of lysozyme has not been elucidated at low pH conditions. Further, the combined effect of sorbitol and GB under low pH conditions is also unexplored. To elucidate the mechanism of action of GB, sorbitol, and their equimolar combinations, we have studied their effect on lysozyme at different concentrations of the additives at pH 2.1, 2.4, 2.7, and 3.2. Lysozyme's thermal denaturation curves were obtained in the presence of different concentrations of sorbitol, glycine betaine, and their equimolar combinations at different pH. In their combined presence, there is no significant enhancement or decrease in the values of thermodynamic parameters including temperature (T1/2), enthalpy of transition (ΔH), constant-pressure heat capacity change (ΔCp), free energy at 25 ℃ (ΔG0), and preferential solvation (ΔΓ23) at different pH conditions which indicates that there is no effective competition or synergy amongst the osmolytes and each one interacts independently within the studied range of osmolyte concentration and pH. Furthermore, fluorescence spectra and CD spectra demonstrated that the secondary and tertiary structure of lysozyme are unperturbed in the case of glycine betaine and sorbitol at different concentrations and pH.