Light Scattering & Flourescence Study of Sugar Solutions as Cryoprotective Agents

光散射

• 批准号: 7691080
• 负责人: DAVID LEE SIDEBOTTOM
• 金额: $ 10.84万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7691080
• 关键词: Annual Reports; Behavior; Biocompatible Materials; Bioinformatics; Biological; Biological Preservation; Books; Carbohydrates; Cells; Cryopreservation; Cryoprotective Agents; Development; Diffuse; Disease; Education; Educational process of instructing; Effectiveness; Electrolytes; Engineering; Evolution; Faculty; Fluorescence; Freeze Drying; Freezing; Frequencies; Gel; Glass; Glucose; Green Fluorescent Proteins; Hydration status; Hydrogen Bonding; Ice; In Situ; Investigation; Knowledge; Lead; Learning; Left; Liquid substance; Literature; Measurement; Measures; Membrane; Membrane Proteins; Modeling; Monitor; Morphology; Motion; Nature; Organism; Outcome; Particle Size; Phase; Phospholipids; Photons; Play; Process; Property; Proteins; Radial; Raffinose; Relaxation; Research; Role; Sampling; Science; Sentinel; Series; Signal Transduction; Solid; Solutions; Solvents; Spectrum Analysis; Staging; Structure; Students; Sucrose; Supervision; Surface; System; Technology; Temperature; Testing; Tissue Engineering; Tissue Preservation; Tissue membrane; Tissues; Transition Temperature; Transplantation; Trehalose; Uncertainty; Universities; Water; Wisconsin; Work; aqueous; chymotrypsin inhibitor; college; human tissue; improved; interest; light scattering; molecular dynamics; protein structure function; scaffold; science education; sugar; theories

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.

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许多生物体的脱水生物中具有显着的作用。已经发展出两种主要理论来解释糖溶液在稳定生物分子中的作用。玻璃化模型只是强调糖使水塑化的性质，从而使其在凝固时形成无定形（玻璃）相（与晶体相反）。通过这种方式，由于周围液体中发生的所有运动被阻止，生物分子（例如蛋白质、磷脂双层）得以稳定。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优先被糖上的氢键置换的结果。键合糖的存在促进了凝固过程中蛋白质功能的保留。此外，最近的分子动力学模拟和我们之前对葡萄糖水溶液的光散射研究强调了这些溶液中糖分子的明显聚集，这些糖分子在高浓度下导致氢键碳水化合物渗透网络的形成。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起什么作用尚不清楚。为了更好地了解凝胶化和玻璃化在生物分子冷冻保存中的双重作用，克赖顿大学的学生将对各种糖浓度下的三种流行的水性糖进行全面的光散射研究，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，在选定的温度下进行，以表征系统中存在的固有动态）。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 和胰凝乳蛋白酶抑制剂 2、CI2）融入这些糖溶液中。两种蛋白质都是荧光的，荧光相关光谱将用于通过观察到这些团聚物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，同时测量荧光产量。在 CI2 中，两种状态转变（自然与变性）通过荧光的显着变化在原位发出信号，并且可用于评估蛋白质的存活是否是（a）猝灭（即玻璃化程度）的函数，（2）凝胶化的函数或（3）糖键引起的水置换的函数。