Hillgren Anna, Lindgren Jan, Aldén Maggie
Department of Pharmacy, Physical and Inorganic Pharmaceutical Chemistry, Uppsala University, Biomedical Center, P.O. Box 580, S-75123 Uppsala, Sweden.
Int J Pharm. 2002 Apr 26;237(1-2):57-69. doi: 10.1016/s0378-5173(02)00021-2.
The purpose of the study was to investigate the protective mechanism of a non-ionic surfactant, Tween 80, at freeze-thawing with controlled temperature history of a model protein, lactate dehydrogenase (LDH). The system was examined by differential scanning calorimetry (DSC) and infrared spectroscopy (IR). LDH activity assays were performed spectrophotometrically. In all samples, independent of temperature history and addition of surfactant, all water was crystallized to polycrystalline ice at temperatures below -20 degrees C. The size and perfection of the ice crystals could be varied by a range of cooling rates giving different degrees of undercooling. At Tween concentrations below the cmc at crystallization, lower concentrations were required at low cooling rates compared to higher cooling rates to protect LDH. Concentrations above cmc of Tween reduced the protection at a cooling rate of 5 degrees C min(-1) and at quenching in N(2)(l). The amount of Tween needed for complete protection correlated to the surface area of the ice crystals at a certain temperature history. Tween 80 protects LDH from denaturation at freeze-thawing by hindering its destructive interaction with the ice crystals. The protective effect might be obtained when Tween molecules compete with the protein for sites on the ice surface. The optimum concentration of Tween needed for complete protection is dependent on the temperature history.
本研究的目的是探究一种非离子表面活性剂吐温80在对模型蛋白乳酸脱氢酶（LDH）进行具有可控温度历程的冻融过程中的保护机制。采用差示扫描量热法（DSC）和红外光谱法（IR）对该体系进行检测。通过分光光度法进行LDH活性测定。在所有样品中，无论温度历程和表面活性剂的添加情况如何，在温度低于-20℃时，所有的水都会结晶为多晶冰。冰晶的大小和完整性可通过一系列冷却速率来改变，这些冷却速率会产生不同程度的过冷度。在结晶时吐温浓度低于临界胶束浓度（cmc）的情况下，与较高冷却速率相比，低冷却速率下保护LDH所需的吐温浓度更低。吐温浓度高于cmc时，在5℃·min⁻¹的冷却速率下以及在液氮淬火时会降低保护效果。在特定温度历程下，完全保护所需的吐温量与冰晶的表面积相关。吐温80通过阻碍LDH与冰晶的破坏性相互作用，在冻融过程中保护其不发生变性。当吐温分子与蛋白质竞争冰表面的位点时，可能会获得保护效果。完全保护所需的吐温最佳浓度取决于温度历程。
