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Title: Effects of osmolytes on the conformational stability and hydrodynamic radii of immunity protein 9 and human serum albumin
Author: Charoenkitpaiboon, Chatuphon
ISNI:       0000 0004 5347 5085
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2014
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Osmolytes affect protein stability through direct interactions with a protein, indirectly by perturbing the properties of the solvent water, and by a combination of these. In this thesis the conformational stability of the colicin E9 immunity protein (Im9) and Human serum albumin (HSA) were determined in the absence and presence of osmolytes (trehalose, sucrose, and glycerol) and their effective hydrodynamic radii measured in order to further explore the mechanism of stabilisation. Urea1/2, the midpoint of the unfolding transition, and GH2O, the free energy of unfolding, were measured in a urea-induced denaturation experiment and detected with fluorescence spectroscopy, and hydrodynamic radii (Rh) were measured with pulsed-field gradient NMR. The unfolding curves of Im9 and HSA are shifted to higher urea concentration so that Urea1/2 and GH2O increased, as the osmolyte concentration was increased indicating that Im9 and HSA are more stable in the presence of osmolytes. The Rh of Im9 and HSA increased in the presence of high concentration of trehalose and sucrose but glycerol produced a reduction. My data support the view that trehalose and sucrose act via a preferential hydration mechanism in which the water layer around the protein increases because osmolytes are excluded from the protein surface. In contrast, glycerol acts by interacting directly with the protein surface, and possibly by penetrating it, which causes the protein to become more compact as its void volume is reduced. This increase in compactness induces stabilisation. In addition, HSA formulations were studied for their stability over 6 months under various conditions using trehalose, sucrose and glycerol as stabilisers instead of acetyltryptophan and sodium octanoate, which are used commercially. However, measurements of HSA esterase-like activity and heme binding, and its aggregation state with polyacrylamide gel electrophoresis and DLS showed the osmolytes cannot stabilize the protein under high storage temperatures.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available