Allison S D, Randolph T W, Manning M C, Middleton K, Davis A, Carpenter J F
School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado, 80262, USA.
Arch Biochem Biophys. 1998 Oct 1;358(1):171-81. doi: 10.1006/abbi.1998.0832.
Limited stability impedes the development of industrial and pharmaceutical proteins. Dried formulations are theoretically more stable, but the drying process itself causes structural damage leading to loss of activity after rehydration. Lyophilization is the most common method used to dry proteins, but involves freezing and dehydration, which are both damaging to protein. We compared an air-drying method to freeze-drying to test the hypothesis that terminal dehydration is the critical stress leading to loss of activity. The secondary structure of air-dried and freeze-dried actin was analyzed by infrared spectroscopy and related to the level of activity recovered from the rehydrated samples. Actin dried by either method in the absence of stabilizers was highly unfolded and the capacity to polymerize was lost upon rehydration. The degree of unfolding was reduced by air-drying or freeze-drying actin with sucrose, and the level of activity recovered upon rehydration increased. The addition of dextran to sucrose improved the recovery of activity from freeze-dried, but not air-dried samples. Dextran alone failed to protect the structure and function of actin dried by either method, indicating that proteins are not protected from dehydration-induced damage by formation of a glassy matrix. In some cases, recovered activity did not correlate directly with the level of structural protection conferred by a particular additive. This result suggests that secondary structural protection during drying is a necessary but not sufficient condition for the recovery of activity from a dried protein after rehydration.
稳定性有限阻碍了工业和药用蛋白质的发展。理论上，干燥制剂更稳定，但干燥过程本身会导致结构破坏，从而在复水后导致活性丧失。冻干是用于干燥蛋白质的最常用方法，但涉及冷冻和脱水，这两者都会对蛋白质造成损害。我们将一种空气干燥方法与冷冻干燥进行了比较，以检验终末脱水是导致活性丧失的关键应激这一假设。通过红外光谱分析了空气干燥和冷冻干燥的肌动蛋白的二级结构，并将其与从复水样品中恢复的活性水平相关联。在没有稳定剂的情况下，通过任何一种方法干燥的肌动蛋白都高度解折叠，并且在复水后聚合能力丧失。用蔗糖对肌动蛋白进行空气干燥或冷冻干燥可降低解折叠程度，并且复水后恢复的活性水平增加。向蔗糖中添加葡聚糖可提高冷冻干燥样品（而非空气干燥样品）的活性恢复率。单独使用葡聚糖无法保护通过任何一种方法干燥的肌动蛋白的结构和功能，这表明蛋白质不会因形成玻璃状基质而免受脱水诱导的损伤。在某些情况下，恢复的活性与特定添加剂赋予的结构保护水平并不直接相关。这一结果表明，干燥过程中的二级结构保护是复水后从干燥蛋白质中恢复活性的必要但不充分条件。
