Effect of Dioxane on the Conformation of Lysozyme at Low pH

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Abstract
We have measured the circular dichroism (CD) spectrum of lysozyme [EC 3.2.1.17] at 25 and 30°C and the viscosity at 25°C in water-dioxane mixtures within the range of apparent pH 2.0-2.4. Above 20% dioxane, three positive bands at 294, 289, and 283 nm and a negative band at 262 nm in the near-ultraviolet region gradually decreased in ellipticity until they were replaced by an overall negative broad spectrum above 30% dioxane. On the other hand, negative CD bands in the far-ultraviolet region with a shoulder at 222 nm and an extremum at 208 nm first decreased slightly in ellipticity at 20-30% dioxane, and then gradually increased up to 40 % dioxane, above which the ellipticity of these bands was still further enhanced. Such profiles were common to the two temperatures studied, but at 30°C a higher resolution of the far-ultraviolet CD bands was observed for lysozyme in 60 and 72% dioxane, suggesting the partial disruption of β-structure. These results indicate that the tertiary structure reflected by the near-ultraviolet CD bands was initially disrupted at 20-30% dioxane with possible slight disorganization of the ordered secondary structure, and then a marked increase in helical structure was induced at high dioxane concentrations. The helical content at 72% dioxane calculated from the ellipticity values at 222 nm was about twice that of lysozyme in the absence of dioxane at both temperatures. The gross structural changes above 20% dioxane were confirmed by the intrinsic viscosities observed at 0, 20, 40, and 60 % dioxane. The ordered structure enhanced by dioxane in the lysozyme molecule was found to be more discernible when the difference CD spectrum was constructed from the observed spectra, taking the CD spectrum of lysozyme in the absence of dioxane as a reference. This procedure was also applied to investigate the effect of pH on the conformation of lysozyme at 60% dioxane. The appearance of β-structure at apparent pH 5.6 and of aggregated helices at apparent pH 7.5 was apparently induced by 60% dioxane.

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