Allison S D, Dong A, Carpenter J F
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
Biophys J. 1996 Oct;71(4):2022-32. doi: 10.1016/S0006-3495(96)79400-6.
Studies of numerous proteins with infrared spectroscopy have documented that unfolding is a general response of unprotected proteins to freeze-drying. Some proteins that are unfolded in the dried solid aggregate during rehydration, whereas others refold. It has been proposed for the latter case that aggregation is avoided because refolding kinetically outcompetes intermolecular interactions. In contrast, with proteins that normally aggregate after rehydration, minimizing unfolding during freeze-drying with stabilizer has been shown to be needed to favor the recovery of native protein molecules after rehydration. The purpose of the current study was to examine first the opposite situation, in which a denaturant is used to foster additional unfolding in the protein population during freeze-drying. If the protein is not intrinsically resistant to aggregation under the study conditions (e.g., because of intermolecular charge repulsion) and the denaturant does not disrupt intermolecular interactions during rehydration, this treatment should favor aggregation upon rehydration. With infrared spectroscopy we found that at concentrations of the denaturant Na thiocyanate (NaSCN) that only slightly perturbed chymotrypsinogen secondary structure in solution before freeze-drying, there was a large increase in protein unfolding in the dried solid and in protein aggregation measured after rehydration. Bands assigned to intermolecular beta sheet were present in the spectra of samples dried with NaSCN, indicating that aggregation could also arise in the dried solid. By examining the protein structure in the frozen state, we determined that in the absence of NaSCN the protein remains native. NaSCN caused structural perturbations during freezing, without the formation of intermolecular beta sheet, that were intermediate to structural changes noted after freeze-drying. In contrast, samples treated in the presence of NaSCN and sucrose had native-like spectra in the frozen and dried states, and much reduced aggregation after rehydration. These results indicate that during freezing and drying the sugar can counteract and mostly reverse the structural perturbations induced by NaSCN before and during these treatments.
利用红外光谱对多种蛋白质进行的研究表明，去折叠是未受保护的蛋白质对冷冻干燥的普遍反应。一些在干燥固体中去折叠的蛋白质在复水过程中会聚集，而另一些则会重新折叠。对于后一种情况，有人提出，由于重新折叠在动力学上胜过分子间相互作用，因此可以避免聚集。相比之下，对于那些在复水后通常会聚集的蛋白质，已证明需要在冷冻干燥过程中使用稳定剂将去折叠程度降至最低，以利于复水后天然蛋白质分子的恢复。本研究的目的是首先研究相反的情况，即在冷冻干燥过程中使用变性剂促使蛋白质群体发生更多的去折叠。如果在研究条件下蛋白质本身对聚集不具有抗性（例如，由于分子间电荷排斥），并且变性剂在复水过程中不会破坏分子间相互作用，那么这种处理应该有利于复水时的聚集。通过红外光谱，我们发现，在冷冻干燥前仅轻微扰动溶液中胰凝乳蛋白酶原二级结构的变性剂硫氰酸钠（NaSCN）浓度下，干燥固体中的蛋白质去折叠以及复水后测得的蛋白质聚集都大幅增加。在用NaSCN干燥的样品光谱中出现了归属于分子间β折叠的谱带，表明聚集也可能在干燥固体中发生。通过检查冷冻状态下的蛋白质结构，我们确定在没有NaSCN的情况下蛋白质保持天然状态。NaSCN在冷冻过程中引起结构扰动，但没有形成分子间β折叠，这种扰动介于冷冻干燥后观察到的结构变化之间。相比之下，在NaSCN和蔗糖存在下处理的样品在冷冻和干燥状态下具有类似天然的光谱，并且复水后的聚集大大减少。这些结果表明在冷冻和干燥过程中，糖可以抵消并在很大程度上逆转NaSCN在这些处理之前和期间引起的结构扰动。
