Yadav Bindu, Chahar Deepak, Kahlon Navjot Kaur, Weber Cameron C, Venkatesu Pannuru
Department of Chemistry, University of Delhi, Delhi 110007, India.
School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland 1010, New Zealand; MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand.
Int J Biol Macromol. 2025 Jul 22;321(Pt 1):146238. doi: 10.1016/j.ijbiomac.2025.146238.
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 (NBr) 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 NBr:E, NBr:H, NBr:O, and NBr:D respectively. Spectroscopic assessment demonstrated that the stability of the Hb structure increased in the order NBr:H < NBr:O < NBr:D < NBr:E, with negligible structural changes observed for Hb in NBr:E even at the highest concentration of exposure. The thermal stability of Hb is reduced for all DESs concentrations (10-30 mg/mL), with NBr:E and NBr: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 NBr:E and hydrophobic NBr:D provide improved stability of Hb over the intermediate polarity NBr:H and NBr:O. These outcomes offer new information for solvent design, towards the stabilisation and use of proteins in alternative media.
非水溶剂在水溶液中用作添加剂时，会影响蛋白质的结构、稳定性和功能，这可能会对纳米技术、生物医学应用产生影响。低共熔溶剂（DESs）是一类有前景的新型非水“绿色溶剂”，有望替代传统溶剂，尽管它们对蛋白质结构和稳定性的影响仍相对了解较少。为了评估DESs中氢键供体（HBD）的烷基链长度对蛋白质结构和胶体稳定性的影响，我们使用氢键受体（HBA）四丁基溴化铵（NBr）合成了四种DESs，并评估了它们对作为模型蛋白的血红蛋白（Hb）的影响。乙醇（E）、己醇（H）、辛醇（O）和癸醇（D）是选择作为HBD用于以1:2（HBA:HBD）摩尔比合成DESs的四种醇。这些DESs在此分别称为NBr:E、NBr:H、NBr:O和NBr:D。光谱评估表明，Hb结构的稳定性按NBr:H < NBr:O < NBr:D < NBr:E的顺序增加，即使在最高暴露浓度下，在NBr:E中观察到的Hb结构变化也可忽略不计。对于所有DESs浓度（10 - 30 mg/mL），Hb的热稳定性均降低，其中NBr:E和NBr:D导致的稳定性降低最小。研究了Hb的胶体稳定性，DESs对Hb单体和聚集体尺寸稳定性的影响具有相似的定性趋势，zeta电位和透射电子显微镜（TEM）分析证实了这一点。这些结果突出表明，Hb的稳定性取决于DES与蛋白质之间亲水和疏水相互作用的相互作用。亲水性更强的NBr:E和疏水性的NBr:D比中等极性的NBr:H和NBr:O能更好地提高Hb的稳定性。这些结果为溶剂设计提供了新信息，有助于在替代介质中稳定和使用蛋白质。
