Stability of beta-galactosidase, a model protein drug, is related to water mobility as measured by 17O nuclear magnetic resonance (NMR).

The inactivation of freeze-dried beta-galactosidase during storage was studied, focusing on the effect of water mobility as measured by the spin-lattice relaxation time, T1, of water using 17O NMR. Inactivation of beta-galactosidase lyophilized from phosphate buffer solution was studied as a function of water content, which in turn affected the T1 of water. An increase in the water content of freeze-dried beta-galactosidase brought about an increase in the T1 of water, as well as a rise in pH. For the freeze-dried enzyme with sufficient water content to be dissolved, the inactivation rate was related to the T1 of water rather than to the pH change. It is suggested that as the water content increases, the mobility of water around the enzyme increases, resulting in enhanced enzyme inactivation. The freeze-dried samples with limited moisture showed inactivation rates faster than those expected from the pH and water mobility, suggesting that the inactivation mechanism is different from that for the freeze-dried enzyme with a larger amount of water. Inactivation of beta-galactosidase in solutions was also studied as a function of phosphate buffer and sodium chloride concentrations, which in turn affected the T1 of water. Because the inactivation rate increased with increasing salt concentrations and the rate extrapolated to zero concentration was negligible, inactivation of the freeze-dried enzyme was apparently induced by the salts used as additives for lyophilization. The enhancing effect of phosphate buffer components, however, was reduced at higher concentrations, an effect related to the decrease in the T1 of water. This result may be ascribed to the decrease in water mobility caused by phosphate buffer components and is consistent with the observation that the inactivation rate of the freeze-dried enzyme with a relatively large amount of water decreased with decreasing T1 of water.