Early stages in the trifluoroethanol-induced unfolding of hen egg-white lysozyme and its complex with (GlcNAc)3.

The trifluoroethanol-induced unfolding of hen egg-white lysozyme was studied by circular dichroism. It was shown that if the H2O/trifluoroethanol ratio is above 10:1 (v/v), the unique three-dimensional structure of the protein is not affected, whereas within the ration 10:1-2.8:1 (v/v), this structure is partially unfolded. At the ratio 2.4:1 (v/v), the native conformation of lysozyme is completely disrupted and the conformational transition fits a two-state model. A similar effect was observed for the trifluoroethanol-induced unfolding of the lysozyme-(GlcNAc)3 complex. Within the H2O2 trifluoroethanol ratio 15:1-5.5:1 (v/v), the characteristic intensities of the Cotton effects which arise from the association of (GlcNAc)3 with the active site of lysozyme, diminished and approached those exhibited by lysozyme itself at the same H2O trifluoroethanol ratios. This shows that (GlcNAc)3 is released from the protein surface in early stages of the unfolding process. At the ratio 2.4:1 (v/v), the lysozyme-(GlcNAc)3 complex was completely disrupted and the protein unfolded. It is suggested that a considerable alteration in hydration of the lysozyme molecule caused by trifluoroethanol increases protein surface fluctuations, causing the release of (GlcNAc)3 from the active site of lysozyme.