Caves, Michael S. (2011) The mechanism of enzyme inactivation and the effects of additives. PhD thesis, University of Warwick.

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Abstract

When kept in aqueous solution at ambient temperature many proteins
undergo denaturation over a relatively short time-scale, leading to loss of
structure and function. The inclusion of small additives, such as polyols and
salts, in protein formulations can be used to inhibit this process, but the
effects of such additives are far from predictable. This thesis details the
biophysical characterisation of the thermal denaturation of three enzymes,
and the effects of three small additives on the stability of these enzymes. The
biophysical characterisation is performed for the most part by circular
dichroism, fluorescence spectroscopy and size exclusion chromatography,
with the results of such studies being correlated with those of activity assays.
All three proteins studied form molten globules upon thermally induced
inactivation, with irreversibility being conferred by aggregation. A little-studied
variant of Glucose Oxidase was found to undergo complete irreversible
inactivation with very little change in secondary structure. NaCl was found to
destabilise this variant, despite previously being shown to stabilise a more
widely studied glucose oxidase.
Uricase was found to undergo significant exposure of hydrophobic residues
to solution upon inactivation, with denaturation occurring over two phases.
NaCl was found to destabilise uricase, while TMAO and glycerol were found
to stabilise the tetramer. However, the presence of TMAO, widely believed to
stabilise protein secondary structure through the osmophobic effect, was
found to alter the structure of the haem-containing active site of horseradish
peroxidase, destabilising this enzyme.
In addition, the effects of NaCl and glycerol on uricase inactivation were
found to change with temperature. In combination, the findings detailed in
this work have implications both for the understanding of enzyme
denaturation, and the value of using the results of additive studies on certain
enzymes under specific conditions to predict the effect that such additives
have on other enzymes under different conditions.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Proteins -- Denaturation, Enzymes -- Additives
Official Date:	September 2011
Dates:	Date Event September 2011 Submitted
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Freedman, R. B.
Sponsors:	Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC) ; Arecor Ltd.
Extent:	xvi, 271 leaves : ill., charts
Language:	eng

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