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

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.

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