Understanding the relationship between deep eutectic solvent polarity and the structural integrity and stability of hemoglobin: A balance between hydrophobicity and hydrophilicity.

Non-aqueous solvents, when used as additives in aqueous solutions, can affect protein structure, stability and function which can have consequences for nanotechnology, biomedical applications. Deep eutectic solvents (DESs) are a promising new category of non-aqueous "green solvents" that may substitute traditional solvents, although their impact on protein structure and stability remains relatively poorly understood. To assess the impact of the alkyl chain length of the hydrogen bond donor (HBD) in DESs on the structural and colloidal stability of proteins, we have synthesized four DESs using the hydrogen bond acceptor (HBA) tetrabutylammonium bromide (N4444Br) and assessed their effect on hemoglobin (Hb) as a model protein. Ethanol (E), hexanol (H), octanol (O), and decanol (D) were the four alcohols chosen as HBD for the synthesis of DESs in 1:2 (HBA: HBD) molar ratios. These DESs are referred to herein as N4444Br:E, N4444Br:H, N4444Br:O, and N4444Br:D respectively. Spectroscopic assessment demonstrated that the stability of the Hb structure increased in the order N4444Br:H < N4444Br:O < N4444Br:D < N4444Br:E, with negligible structural changes observed for Hb in N4444Br:E even at the highest concentration of exposure. The thermal stability of Hb is reduced for all DESs concentrations (10-30 mg/mL), with N4444Br:E and N4444Br:D resulting in the smallest decrease in stability. Hb colloidal stability was investigated, with similar qualitative trends in the DES impacts on the stability of Hb monomer and aggregate sizes, corroborated by zeta-potential and TEM analyses. These results highlight that Hb stability is dependent on the interplay between hydrophilic and hydrophobic interactions between the DES and the protein. The more hydrophilic N4444Br:E and hydrophobic N4444Br:D provide improved stability of Hb over the intermediate polarity N4444Br:H and N4444Br:O. These outcomes offer new information for solvent design, towards the stabilisation and use of proteins in alternative media.