Light Scattering & Flourescence Study of Sugar Solutions as Cryoprotective Agents

糖水溶液在生物膜和可溶性蛋白质的冷冻保存和干燥以及自然界中许多生物体的脱水过程中具有显著的特征。两种主要的理论已经发展来解释糖溶液在稳定生物分子中的作用。玻璃化模型简单地强调了糖对水的溶解性，从而使其在固化时形成无定形（玻璃状）相（与晶体相反）。以这种方式，生物分子（例如，蛋白质、磷脂双层）由于阻止了周围液体中发生的所有运动而稳定。相比之下，水置换模型提出，生物分子的稳定性是蛋白质附近的水如何优先被与糖的氢键取代的结果。键合糖的存在促进了蛋白质功能在固化过程中的保留。此外，最近的分子动力学模拟和我们自己以前的光散射研究的葡萄糖水溶液突出了显着的集群的糖分子在这些解决方案中，在高浓度下导致形成的氢结合的碳水化合物的一个heterogenated网络。这种凝胶网络显然有助于溶液的整体玻璃化，但它在生物分子表面附近的水置换中扮演什么角色尚不清楚。为了更好地了解凝胶化和玻璃化在生物分子低温保存中的双重作用，Creighton University的学生将对三种常见的水性糖进行一系列全面的光散射研究，这些糖浓度范围很广，以正确表征这些增稠液体中存在的固有结构和动力学。该研究将包括静态光散射（表征系统中存在的固有结构）和动态光散射（光子相关光谱，在选定温度下进行，以表征系统中存在的固有动力学）。在我们项目的第二阶段，学生将把功能性生物分子（绿色荧光蛋白，GFP和糜蛋白酶抑制剂2，CI2）纳入这些糖溶液。这两种蛋白质都是荧光的，荧光相关光谱将用于通过观察到的这些聚集体在溶液中扩散时的流体动力学半径的变化来确定糖与蛋白质的结合程度。然后将样品淬灭至不同的玻璃化状态，同时测量荧光产率。在C12中，通过荧光的显著变化原位发出两态转变（天然与变性）的信号，并且可以用于评估蛋白质的存活是否是（a）猝灭的函数（即，玻璃化程度），（2）胶凝作用或（3）由糖键合引起的水置换作用。

Aqueous sugar solutions feature prominently in the cryopreservation and drying of biological membranes and soluable proteins as well as in the anhydrobiosis of many organisms in nature. Two dominant theories have developed to explain the role of sugar solutions in stabilizing a biomolecule. The vitrification model simply emphasizes the nature of sugars to plasticize water, thus allowing it to form an amorphous (glassy) phase (as opposed to crystal) when solidified. In this way the biomolecule (e.g., protein, phospholipid bilayer) is stabilized as a consequence of the arrest of all motions occurring in the surrounding liquid. By contrast, the water replacement model proposes that stability of the biomolecule is a result of how water near the protein is preferentially replaced by hydrogen bonding to the sugar. The presence of the bonded sugar promotes retention of protein functionality during solidfication. In addition, recent molecular dynamics simulations and our own previous light scattering studies of aqueous glucose solutions highlight pronounced clustering of sugar molecules in these solutions which at high concentrations lead to the formation of a percolated network of hydogen bonded carbohydrates. This gel network clearly aids in the overall vitrification of the solution, but what role it plays in water replacement near the surface of a biomolecule is yet unknown. To better understand the dual roles of gelation and vitrification in the cryopreservation of biomolecules, students at Creighton University will conduct a comprehensive set of light scattering investigations of three popular aqueous sugars over a wide range of sugar concentrations to properly characterize the inherent structures and dynamics present in these thickening liquids. The study will include both static light scattering (characterizing inherent structures present in the system) and dynamic light scattering (photon correlation spectroscopy, performed at selected temperatures to characterize inherent dynamics present in the system). In a second phase of our project, students will incorporate functional biomolecules (green fluorescent protein, GFP and chymotrypsin inhibitor 2, CI2) into these sugar solutions. Both proteins are fluorescent and fluorescence correlation spectroscopy will be used to determine the degree of bonding of sugars to the protein through changes observed in the hydrodynamic radius of these agglomerates as they diffuse in the solution. Samples will then be quenched to differing states of vitrification while the fluorescence yield is measured. In CI2 the two-state transition (natured vs. denatured) is signaled in situ by a pronounced change in fluorescence and can be used to evaluate whether the survival of the protein is either (a) a function of the quenching (i.e., degree of vitrification), (2) a function of gelation or (3) a function of water replacement caused by sugar bonding.