Solvation of lysozyme in water/dioxane mixtures studied in the frozen state by NMR spectroscopy

Abstract

The amounts of water and dioxane bound to lysozyme (EC 3.2.1.17) in acidic aqueous dioxane mixtures were investigated in the frozen state by nuclear magnetic resonance spectroscopy. Hydration was measured using a mixed solvent, water/[U- 2H]dioxane; dioxane solvation was measured using a mixed solvent, dioxane/ 2H 2O. The effect of temperature on the proton NMR spectra of solvents bound to lysozyme was examined, and −35 °C was selected as an appropriate temperature for the NMR measurements. The lysozyme molecule was found to bind about 0.30 g of water/g of lysozyme in an aqueous solution, and this value lies between those obtained by other methods. With increasing dioxane concentration the amount of hydration first decreased to 0.2 g/g at 10% dioxane, and then leveled off. An increase in dioxane concentration from 40 to 58% induced a slight increase in the amount of hydration. The amount of dioxane solvation of lysozyme first showed a gradual decrease with increase in dioxane concentration to 40%, then it increased rapidly to 0.7 g/g at 57% dioxane concentration. This change in the amount of solvation with dioxane concentration approximately parallels the increase of α-helical content. The interaction of the solvent components with lysozyme is discussed in relation to the protein conformation.