Unveiling the Three-Step Model for the Interaction of Imidazolium-Based Ionic Liquids on Albumin

[Image: see text] The effect of the ionic liquids (ILs) 1-methyl-3-tetradecylimidazolium chloride ([C(14)MIM][Cl]), 1-dodecyl-3-methylimidazolium chloride ([C(12)MIM][Cl]), and 1-decyl-methylimidazolium chloride ([C(10)MIM][Cl]) on the structure of bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, ultraviolet–visible (UV–vis) spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations. Concerning the fluorescence measurements, we observed a blue shift and a fluorescence quenching as the IL concentration increased in the solution. Such behavior was observed for all three studied imidazolium-based ILs, being larger as the number of methylene groups in the alkyl chain increased. UV–vis absorbance measurements indicate that even at relatively small IL/protein ratios, like 1:1 or 1:2, ([C(14)MIM][Cl]) is able to change, at least partially, the sample turbidity. SAXS results agree with the spectroscopic techniques and suggest that the proteins underwent partial unfolding, evidenced by an increase in the radius of gyration (R(g)) of the scattering particle. In the absence and presence of ([C(14)MIM][Cl]) = 3 mM BSA R(g) increases from 29.1 to 45.1 Å, respectively. Together, these results indicate that the interaction of BSA with ILs is divided into three stages: the first stage is characterized by the protein in its native form. It takes place for protein/IL ≤ 1:2, and the interaction is predominantly due to the electrostatic forces provided by the negative charges on the surface of BSA and the cationic polar head of the ILs. In the second stage, higher IL concentrations induce the unfolding of the protein, most likely inducing the unfolding of domains I and III, in such a way that the protein’s secondary structure is kept almost unaltered. In the last stage, IL micelles start to form, and therefore, the interaction with protein reaches a saturation point and free micelles may be formed. We believe that this work provides new information about the interaction of ILs with BSA.