The influence of glycerol on hydrogen isotope exchange in lysozyme

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Abstract
Hydrogen isotope exchange rates for lysozyme in glycerol cosolvent mixtures [D. G. Knox and A. Rosenberg (1980) Biopolymers 19, 1049–1068] have been analyzed as functions of solvent viscosity and glycerol activity in an attempt to determine which solvent properties influence protein internal dynamics. The effect of glycerol on the fast‐ and slow‐exchanging protons is different. Slow‐exchanging protons [H(t) < 20] are slowed by ever‐increasing amounts as H(t) decreases. However, comparison with data for the effect of glycerol on the thermal unfolding of lysozyme [K. Gekko (1982) J. Biochem. 19, 1197–1204] indicates that the large decrease in exchange rates for the slow protons is not consistent with a local unfolding mechanism of exchange. These effects are also too large to be easily rationalized in terms of solvent viscosity. Instead, we suggest that the large effect of glycerol on exchange of the slow protons is due to a “compression” of the protein, as a result of thermodynamically unfavorable interactions of glycerol with the protein surface. This reduces the protein void volume, which in turn decreases the probability of conformational transitions required for exchange of the slowest protons. Present data do not allow a distinction to be made between thermodynamic (glycerol activity) and dynamic (solvent viscosity) influences on exchange rates for the fast‐exchanging protons, although the effect of glycerol on these protons is also probably too large to be consistent with a local unfolding mechanism. In this case, glycerol decreases the rate of catalyst diffusion within the protein matrix, either by decreasing the probabilities or amplitudes of “gating” reactions that allow passage of the catalyst from the solvent to the exchange site, or by increasing the relaxation times for these conformational rearrangements.