Effect of an ionic liquid on the unfolding of human serum albumin: a fluorescence correlation spectroscopy study

Abstract

The effect of the room temperature ionic liquid (RTIL) 1-pentyl-3-methyl-imidazolium bromide ([pmim][Br]) on the unfolding of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). The structural fluctuations of the protein exhibit three characteristic time constants, namely, ~3, ~35 and ~260μs. On addition of the RTIL, the dynamics become slightly slower, with time constants of ~5, ~40 and ~350μs. The two fast components (3 and 35μs in the absence of RTIL and 5 and 40 μ s in the presence of RTIL) are assigned to chain motion of the protein. The slowest component (260 or 350 μ s) may arise from detachment (unbinding) of the non-covalent dye from the protein. In the absence of RTIL-and on addition of guanidinium hydrochloride (GdnHCl)-as the protein unfolds, the contribution of the fastest component increases rapidly from 10% at 1M to 40% at 6m, and its time constant decreases from 3μs to 1μs. In the presence of RTIL, the addition of GdnHCl causes significant changes in both the structure (CD spectrum) and the time constants of conformational fluctuation. In the presence of the RTIL, the addition of GdnHCl gives rise to a very slow component (1025 μs in 1M and 560μs in 6M GdnHCl). It is proposed that the guanidinium cation (GdnH⁺) repels the imidazolium cation ([pmim]⁺) at the protein surface, and this causes a change in the structure and dynamics of the protein. On addition of 6M GdnHCl, the diffusion coefficient of C153 bound to HSA decreases. The hydrodynamic radius of the denatured protein (in 6M GdnHCl) is larger than that of the native protein (about 1.75 times in the absence of RTIL and 2.6 times in the presence of RTIL).