Light Scattering & Flourescence Study of Sugar Solutions as Cryoprotective Agents

糖水溶液在生物膜和可溶蛋白质的超低温保存和干燥以及自然界中许多生物的脱水生物中具有突出的作用。两种占主导地位的理论已经发展起来，用来解释糖溶液在稳定生物分子方面的作用。玻璃化模型简单地强调了糖对水塑化的性质，从而使水在凝固时形成无定形(玻璃状)相(而不是晶体)。以这种方式，生物分子(例如，蛋白质、磷脂双层)由于周围液体中发生的所有运动的停止而稳定。相比之下，水替代模型提出，生物分子的稳定性是蛋白质附近的水优先被糖上的氢键取代的结果。结合糖的存在促进了蛋白质在固化过程中的功能保留。此外，最近的分子动力学模拟和我们以前对葡萄糖水溶液的光散射研究强调了糖分子在这些溶液中明显的聚集，这在高浓度时导致形成一个渗入的氢键碳水化合物网络。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和玻璃化在生物分子超低温保存中的双重作用，克雷顿大学的学生将对三种常见的水糖在广泛的糖浓度范围内进行一套全面的光散射研究，以适当地表征这些增稠液体中存在的内在结构和动力学。这项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，在选定的温度下执行，以表征系统中存在的固有动力学)。在我们项目的第二阶段，学生们将把功能性生物分子(绿色荧光蛋白，GFP和胰凝乳杆菌抑制物2，CI2)加入到这些糖溶液中。这两种蛋白质都是荧光的，荧光相关光谱将通过观察到这些凝聚体在溶液中扩散时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，样品将被淬灭到不同的玻璃化状态，同时测量荧光产量。在CI2中，双态转变(自然的和变性的)是通过荧光的显著变化在原位发出信号的，可以用来评估蛋白质的存活率是(A)猝灭(即玻璃化程度)的函数，(2)凝胶的函数，还是(3)糖键引起的水替代的函数。

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.