Locating the binding domains of lysozyme with ionic liquids in aqueous solution via spectroscopic studies Yun Guo a,1 , Bo Zhang a,1 , Chao Lu a,1 , Xiaoxue Liu a , Qing Li a , Hua Zhang b, ⁎, Zhanzhong Wang a, ⁎ a School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China b Chongqing Institute for Food and Drug Control, Chongqing 401121, PR China abstract article info Article history: Received 5 December 2018 Received in revised form 13 February 2019 Accepted 13 February 2019 Available online 14 February 2019 The binding domains of lysozyme with ionic liquids (ILs, [C 4 mim]BF 4 , [C 4 mim]Cl, [C 4 mim]Br and [dmim]I) in aqueous solution was investigated by studying molecular interactions using spectroscopic techniques. Ultraviolet spectroscopy (UV) showed that the addition of ILs increased the absorption peak intensity of lysozyme at 210 nm by enhancing peptide bond valence electron transition. It is also found that a weak interaction between ILs and lysozyme chromophore groups was generated by analyzing the changes of absorption peak intensity near 280 nm. Fluorescence and Synchronous Fluorescence spectra results showed that four ILs had quenching effect on the fluorescent substances of lysozyme, and the quenching effect rose with increasing ILs concentration. Meanwhile, the interaction between lysozyme and ILs molecules is mainly based on Van der Waals force and two Tryptophan (Trp) residues (Trp62 or Trp108) at the active site of lysozyme molecules play a critical role in binding ILs to their own molecules. © 2019 Published by Elsevier B.V. Keywords: Lysozyme Ionic liquids Binding domains Spectroscopic studies 1. Introduction Studies on the interactions between small molecules and bio- macromolecules have attracted increasing attention due to their signif- icant theoretical [1,2] and practical [3,4] implications on molecular biol- ogy [5]. ILs, one class of small molecules, remain in the liquid state for a wide range of temperature [6] and bear remarkable properties like neg- ligible vapor pressure [7] and high electrical conductivity [8]. ILs have been established as an environmental friendly alternative to the con- ventional solvents [9] and are now being largely used as co-solvents [10] or even as reaction media [11] for many bio-macromolecules. Moreover, ILs offer unique environment that can be tuned to alter the structural and biochemical properties of biomacromolecules [12]. A plethora of evidence shows that ILs can alter the stability and func- tions of proteins. For example, the use of 1,3-butylimidazolium chloride, an IL, as an additive was reported to improve the crystallization of lyso- zyme by giving rise to well-shaped larger crystals with lesser poly- morphs [13]. ILs were also recently shown to have a huge effect on the activity, stability, and reusability of immobilized cellulose [14]. Our previous research shows that ILs can influence the intramolecu- lar interactions within the lysozyme molecule in solution, aid the inter- molecular self-assembly and further enhance activity [15,16]. Recently, it has been reported that a particular IL can interact differently with different, or even closely related bio-macromolecules [17–20]. Tseng [21] reported an affinity IL based on biomolecular recognition and found that this IL was capable of quantitative partitioning of biomacromolecules from aqueous buffer to IL. Geng [22] studied the in- teraction of a surface active IL (1-tetradecyl-3-methylimidazolium bro- mide) with bovine serum albumin (BSA). The results showed that this IL binds to BSA by electrostatic interaction at the critical aggregation con- centration and by hydrophobic interaction at the critical micelle con- centration. Nevertheless, investigations on interaction between ILs and bio-macromolecules are still limited and there is an urgent necessity of figuring out the mechanism of interactions of ILs with bio- macromolecules. The structure of hydrogen bond network widely exists in ILs [23]. Due to the existence of such a structure, ILs have the network structure of periodic distribution, showing the characteristics of “liquid molecular sieve” [24]. Bymes achieves different effects by adding differ- ent kinds of ILs to the protein solution, such as improving the thermody- namic stability of proteins, promoting protein folding and denaturation and aggregation of proteins [25]. In this work, Ultraviolet, Fluorescence and Synchronous Fluores- cence Spectrum were used to probe the interaction mechanism be- tween lysozyme molecules and ILs, aiming at illustrating the binding domains and the interaction of lysozyme with ILs, 1-butyl-3- methylimidazolium tetra fluoroborate ([C 4 mim]BF 4 ), 1-butyl-3- methylimidazolium chloride ([C 4 mim]Cl), 1-butyl-3- methylimidazolium bromide ([C 4 mim]Br), and 1,3- dimethylimidazolium iodine ([dmim]I). This work could offer signifi- cant explanation for ILs improving biomacromolecular assembly and Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 214 (2019) 239–245 ⁎ Corresponding authors. E-mail addresses: zhanghua@cqifdc.org.cn (H. Zhang), wzz7698@tju.edu.cn (Z. Wang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.saa.2019.02.032 1386-1425/© 2019 Published by Elsevier B.V. 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