Effects of drying methods and additives on structure and function of actin: Mechanisms of dehydration-induced damage and its inhibition
Effects of drying methods and additives on structure and function of actin: Mechanisms of dehydration-induced damage and its inhibition
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DOI：
10.1006/abbi.1998.0832
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发表时间：
1998-10-01
影响因子：
3.9
通讯作者：
Carpenter, JF
中科院分区：
文献类型：
--
作者：
Allison, SD;Randolph, TW;Carpenter, JF
有限的稳定性阻碍了工业和制药蛋白质的发展。干燥制剂理论上更稳定，但干燥过程本身会导致结构损伤，导致再水化后活性丧失。冻干是用于干燥蛋白质的最常用方法，但涉及冷冻和脱水，这两者都对蛋白质有损害。我们比较了空气干燥法和冷冻干燥法，以检验终末脱水是导致活性丧失的关键应力这一假设。空气干燥和冷冻干燥的肌动蛋白的二级结构进行了分析，通过红外光谱和相关的活性水平回收的再水化样品。在不存在稳定剂的情况下，通过任一方法干燥的肌动蛋白高度展开，并且在再水化后失去了折叠能力。通过空气干燥或冷冻干燥肌动蛋白与蔗糖，展开程度降低，并且在再水化后恢复的活性水平增加。向蔗糖中加入葡聚糖改善了冻干样品的活性恢复，但对风干样品没有改善。单独的葡聚糖不能保护肌动蛋白的结构和功能，这表明蛋白质不能通过形成玻璃状基质来保护其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没有直接关系。这一结果表明，在干燥过程中的二级结构保护是一个必要的，但不是充分的条件，从干燥的蛋白质再水化后的活性恢复。(C)北京：科学出版社.
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. (C) 1998 Academic Press.