Yoshioka S, Aso Y, Izutsu K, Terao T
National Institute of Hygienic Sciences, Tokyo, Japan.
Pharm Res. 1993 Jan;10(1):103-8. doi: 10.1023/a:1018933315538.
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.
研究了冻干β-半乳糖苷酶在储存过程中的失活情况，重点关注通过利用17O NMR测量水的自旋晶格弛豫时间T1来衡量的水流动性的影响。研究了从磷酸盐缓冲溶液冻干的β-半乳糖苷酶的失活情况与水分含量的关系，而水分含量又会影响水的T1。冻干β-半乳糖苷酶水分含量的增加导致水的T1增加，同时pH值也升高。对于水分含量足以溶解的冻干酶，失活速率与水的T1相关，而不是与pH变化相关。有人提出，随着水分含量增加，酶周围水的流动性增加，导致酶失活增强。水分有限的冻干样品的失活速率比根据pH值和水流动性预期的要快，这表明失活机制与水分含量较高的冻干酶不同。还研究了溶液中β-半乳糖苷酶的失活情况与磷酸盐缓冲液和氯化钠浓度的关系，而这又会影响水的T1。由于失活速率随着盐浓度的增加而增加，并且外推到零浓度时的速率可以忽略不计，冻干酶的失活显然是由用作冻干添加剂的盐引起的。然而，磷酸盐缓冲液成分的增强作用在较高浓度下会降低，这一作用与水的T1降低有关。这一结果可能归因于磷酸盐缓冲液成分导致的水流动性降低，并且与观察到的水分含量相对较高的冻干酶的失活速率随着水的T1降低而降低的现象一致。
