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It is thought that there is little or no change in globular protein conformation upon removal of solvent water. This idea is supported by a number of ~t u d i e s~,~,~ on lysozyme which are consistent with a constant conformation as the protein is taken from aqueous solution to complete dryness. We now report a difference hydrogen-exchange study in which lyophilized lysozyme samples, either completely dry or hydrated a t a high relative humidity, were compared with lysozyme in solution. The results provide strong, though indirect, evidence that lysozyme in lyophilized samples cannot have the same conformation as it does in aqueous solution.Solutions of hen egg-white lysozyme sulfate were prepared by dissolving salt-free lysozyme in water, passing the solution through an anion exchange column in the sulfate form, and adjusting the pH to 4.0 with dilute sulfuric acid. Samples of these solutions were freeze-dried to complete dryness4 and kept a t 20OC. Equilibration at 94% relative humidity was achieved by exposing evacuated samples to water vapor from a saturated solution of potassium nitrate.5 In some cases these rehydrated lyophilized samples were completely dried a second time.In the hydrogen-exchange measurements, deionized tritiated water was added to a lyophilized sample for a 3-min pulse of tritium exchange at 20°C. The protein concentration was 1% and the pH remained at 4.0. After the 3-min period, the solution was transferred to a rapid-dialysis apparatus: and the tritiated water removed in the presence of 0.1M acetate buffer at pH 4.0 and 20°C. After the 30 min required for removal of the tritiated water, the exchange out of tritium remaining in the protein was followed for several hours.6 Experiments in which lysozyme in the original solution was pulse-labeled and dialyzed under identical conditions were also done. As a necessary control, experiments were carried out in which lyophilized samples were dissolved in water l /~ hr before pulse labeling.Representative results for tritium exchange out with different lysozyme samples are presented in Fig. 1, where hcdcd, the apparent number of hydrogens per protein molecule exchanged in during the pulse labeling and not yet exchanged out during dialysis, is plotted as a function of time of dialysis. Curve A was obtained for a lysozyme sample in solution at the start of the pulse-labeling period. A slow exchange out of one hydrogen per protein molecule can be seen. This small amount of residual tritium labeling is clearly a net effect due to fractional labeling of a number of slowly exchanging peptide hydrogens during the 3-min exchange in period. Curve B shows data obtained when tritiated water was added to completely dry lysozyme at the start of the 3-min pulse. When lyophilized lysozyme was equilibrated at 94% relative humidity a t 2OoC for 38 hr before labeling, curve C was obtained. Results for a sample that was completely dried, equilibrated a t 94%

SynopsisSorption isotherms of water by proteins show hysteresis that appears to be due to interactions at the molecular level. Four thermodynamically consistent models for this irreversible process are presented. Hysteresis could be the result of slow, incomplete conformational changes occurring upon addition and removal of water. Conformational hysteresis would occur if a number of different conformations, each corresponding to a local free energy minimum, could be present at each pressure of water vapor. Hysteresis might result from an incomplete process of intermolecular phase annealing. Finally, hysteresis might be due to incomplete phase change if two different protein phases are present. Experimental tests for these models are presented. Further study should lead to more insight into the effects of the presence of water on protein conformation and dynamics.

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