Light Scattering & Flourescence Study of Sugar Solutions as Cryoprotective Agents

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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.