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Thermal inactivation of uricase (urate oxidase) : mechanism and effects of additives
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Caves, Michael S., Derham, Barry K., Jezek, Jan and Freedman, R. B. (2013) Thermal inactivation of uricase (urate oxidase) : mechanism and effects of additives. Biochemistry, Volume 52 (Number 3). pp. 497-507. doi:10.1021/bi301334w ISSN 0006-2960.
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Official URL: http://dx.doi.org/10.1021/bi301334w
Abstract
Uricase (Urc) is an oxidoreductase enzyme of both general and commercial interest, the former because of its lack of a cofactor and the latter because of its use in the treatment of hyperuricemic disorders. Results of fluorometry and circular dichroism (CD) spectroscopy indicate that the main phase of thermal Urc inactivation follows an irreversible two-state mechanism, with loss of 20% of the helical structure, loss of the majority of the tertiary structure, and partial exposure of tryptophan residues to solution being approximately concurrent with activity loss. Results of size exclusion chromatography and 8-anilinonaphthalene-1-sulfonate binding studies confirm that this process results in the formation of aggregated molten globules. In addition to this process, CD studies indicate the presence of a rapid reversible denaturation phase that is not completely coupled to the main phase. Urc inactivation is inhibited by the presence of glycerol and trimethylamine oxide, stabilizers of hydrophobic interactions and backbone structure respectively, confirming that loss of hydrophobic bonding and loss of helical structure are key events in the loss of Urc activity. NaCl, however, destabilizes the enzyme at elevated temperature, emphasizing the importance of ionic interactions to Urc stability. A model is developed in which interfacial disruption, involving local loss of hydrophobic interactions, ionic bonds, and helical structure, leads to Urc inactivation and aggregation. Additional studies of Urc inactivation at a more ambient temperature indicate that the inactivation process followed under such conditions is different from that followed at higher temperatures, highlighting the limitations of high-temperature enzyme stability studies.
Item Type: | Journal Article | ||||
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||
Journal or Publication Title: | Biochemistry | ||||
Publisher: | American Chemical Society | ||||
ISSN: | 0006-2960 | ||||
Official Date: | 22 January 2013 | ||||
Dates: |
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Volume: | Volume 52 | ||||
Number: | Number 3 | ||||
Page Range: | pp. 497-507 | ||||
DOI: | 10.1021/bi301334w | ||||
Status: | Peer Reviewed | ||||
Publication Status: | Published | ||||
Access rights to Published version: | Restricted or Subscription Access |
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