Effect of Dioxane on the Conformation of Lysozyme at Low pH1

Abstract

Ultraviolet difference absorption spectra of lysozyme [EC 3.2.1.17] at pH2.1 produced by dioxane were measured as a function of dioxane concentration at 25°C. At 20–30% dioxane an abrupt decrease in molar difference absorption coefficients (Δɛ) was observed in plots of Δɛ₂₉₂ and Δɛ₂₈₅. dioxane concentration; this reflects an unfolding process of lysozyme by dioxane. The magnitude of the decrease in Δε₂₉₂ indicated the exposure of at least one buried tryptophyl residue to the solvent environment in this unfolding process. On the other hand, two linear portions were observed at 0–12% and 40–70% dioxane in the plots, corresponding to solvent effects of dioxane on native and denatured lysozyme, respectively. The number of exposed tryptophyl and tyrosyl residues in these dioxane concentration regions was estimated using the method described previously (Izumi & Inoue (1976) J. Biochem. 70, 1309-1321). The results showed that 3.9 tryptophyl and 1.5 tyrosyl residues were exposed at 0–12%, dioxane while 3.1 tryptophyl and 2.5 tyrosyl residues were exposed at 40–70% dioxane. Tryptophyl and tyrosyl exposures of denatured lysozyme at dioxane concentrations above 40% were also estimated on the basis of difference spectra obtained using a lysozyme solution containing 40% dioxane as a reference. In good agreement with the above results, this modified method gave an exposure of 3.4 tryptophyl and 2.3 tyrosyl residues for denatured lysozyme and some advantages of its use for denatured proteins are discussed. These results indicate that the tryptophyl behavior in denaturation is different from that of tyrosyl residues: the tyrosyl exposure is simply increased upon denaturation, while the tryptophyl exposure is increased in the unfolding process at 20–30% dioxane, and then decreased even below that in the native state above 40% dioxane. The decrease in tryptophyl exposure was attributed to intramolecular reorganization of lysozyme (with the disulfide bridges situated near the tryptophyl residues remaining intact) in the direction of a more helical conformation at higher dioxane concentrations. Fluorescence spectra were also measured for lysozyme in aqueous dioxane mixtures at apparent pH 2.1–2.3 and 25°C. The results were consistent with the unfolding process observed by difference spectral measurements, but at high dioxane concentrations the fluorescence spectra of lysozyme behaved in a complicated manner. This appeared to reflect the state of the tryptophyl residues in relation to the conformational changes in lysozyme. Preliminary results on the sedimentation velocity behavior of lysozyme are also reported.