EFFECTS OF OSMOLYTES ON THE CONFORMATIONAL STABILITY AND HYDRODYNAMIC RADII OF IMMUNITY PROTEIN 9 AND HUMAN SERUM ALBUMIN

Charoenkitpaiboon, Chatuphon (2014) EFFECTS OF OSMOLYTES ON THE CONFORMATIONAL STABILITY AND HYDRODYNAMIC RADII OF IMMUNITY PROTEIN 9 AND HUMAN SERUM ALBUMIN. Doctoral thesis, University of East Anglia.

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Abstract

Abstract
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.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Pharmacy
Depositing User:	Users 2259 not found.
Date Deposited:	06 Mar 2014 11:27
Last Modified:	06 Mar 2014 11:27
URI:	https://ueaeprints.uea.ac.uk/id/eprint/47952
DOI:

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