Chang B S, Kendrick B S, Carpenter J F
Department of Pharmaceutics and Drug Delivery, Amgen, Inc., Amgen Center, Thousand Oaks, CA 91320, USA.
J Pharm Sci. 1996 Dec;85(12):1325-30. doi: 10.1021/js960080y.
In this study, we found that the denaturation of proteins during freezing is closely related to surface-induced denaturation. Several proteins with varying sensitivities to freezing were tested, and the results were compared with susceptibilities to surface denaturation in unfrozen aqueous solution. Also, the influence of the surfactant Tween 80 on the denaturation of each of the proteins was examined during freeze-thawing, as were the effects of Tween 80 and several other surfactants on the stability of lactate dehydrogenase. Proteins formed insoluble precipitates when they were subjected to a quench cooling by dipping in liquid nitrogen, although freezing followed by supercooling caused less precipitation. A strong correlation (r = 0.99) was observed between the tendency of a protein to freeze denature and its tendency to surface denature. Also, the addition of small amounts of surface-active agents protected proteins from both freeze- and surface-induced denaturation. Freeze-induced denaturation of IL-1ra at the ice-water interface during freeze-drying was effectively prevented by adding a small amount of Tween 80. These results suggest that the denaturation of proteins during freeze-thawing can be ascribed primarily to the increase in the area of the ice-water interface during freezing.
在本研究中，我们发现蛋白质在冷冻过程中的变性与表面诱导变性密切相关。测试了几种对冷冻敏感性不同的蛋白质，并将结果与在未冷冻水溶液中对表面变性的敏感性进行了比较。此外，还研究了表面活性剂吐温80在冻融过程中对每种蛋白质变性的影响，以及吐温80和其他几种表面活性剂对乳酸脱氢酶稳定性的影响。当蛋白质通过浸入液氮进行骤冷时会形成不溶性沉淀，不过先冷冻后过冷导致的沉淀较少。观察到蛋白质冷冻变性的倾向与其表面变性的倾向之间存在很强的相关性（r = 0.99）。此外，添加少量表面活性剂可保护蛋白质免受冷冻和表面诱导的变性。通过添加少量吐温80可有效防止冻干过程中冰 - 水界面处白细胞介素 - 1受体拮抗剂（IL - 1ra）的冷冻诱导变性。这些结果表明，蛋白质在冻融过程中的变性主要可归因于冷冻过程中冰 - 水界面面积的增加。
