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dc.contributor.advisorKettle, Tony
dc.contributor.advisorHampton, Mark
dc.contributor.authorHamzah, Melanie Rosina
dc.date.available2015-11-11T21:11:14Z
dc.date.copyright2015
dc.identifier.citationHamzah, M. R. (2015). Inactivation of a Thiol-Dependent Enzyme by Urate Hydroperoxide (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/6064en
dc.identifier.urihttp://hdl.handle.net/10523/6064
dc.description.abstractThere are links between high serum urate (hyperuricemia) and many inflammatory diseases, yet the mechanism is obscure. Urate, the product of purine and ATP break down, builds up in plasma because humans lack the enzyme uricase to convert it to allantoin, which is freely excreted. Urate may benefit health by acting as an antioxidant that scavenges reactive oxygen species. However, hyperuricemia is associated with gout, metabolic syndrome and cardiovascular disease. Oxidative stress is also associated with all these inflammatory diseases. During oxidative stress urate is converted to several reactive electrophiles, including urate hydroperoxide. This novel oxidant could contribute to the adverse effects of urate. Urate hydroperoxide is formed when urate is oxidized to a radical that subsequently combines with superoxide. Activated white blood cells called neutrophils, and xanthine oxidase along with myeloperoxidase/lactoperoxidase, can produce urate hydroperoxide. Previous studies characterized the formation of urate hydroperoxide and its oxidation of small biomolecules. In this investigation, I explored oxidation of thiols and the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by urate hydroperoxide. The effectiveness of urate hydroperoxide as a thiol oxidant was compared with taurine chloramine. Ellman’s assay for reduced thiols was used to measure depletion of cysteine residues on GAPDH by urate hydroperoxide and taurine chloramine. GAPDH was exposed to oxidants in a dose-dependent manner, then assayed by measuring its ability to catalyse the production of NADH. Mass spectrometry was used to identify specific modifications of GAPDH. Urate hydroperoxide oxidized exposed thiols on GAPDH and fully inactivated the enzyme at a ratio of about 5:1. Half of its activity was recovered by reduction with DTT. In comparison, taurine chloramine inactivated GAPDH at approximately 10:1 and DTT reduction recovered all activity. Hence, urate hydroperoxide inactivates GAPDH by reversible and irreversible routes. GAPDH increased in molecular mass by 132 Da with exposure to urate hydroperoxide, indicating the formation of a GAPDH-urate adduct. However, I could not identify which residue was modified with a tryptic digest. Formation of urate hydroperoxide during inflammation and its subsequent oxidative reactions may explain some of the adverse effects of hyperuricemia.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectUrate Hydroperoxide
dc.subjectHyperuricemia
dc.subjectPro-oxidant
dc.subjectMyeloperoxidase
dc.subjectLactoperoxidase
dc.subjectUrate
dc.subjectUric Acid
dc.subjectUrate Adduct
dc.subjectGlyceraldehyde-3-phosphate dehydrogenase
dc.subjectGAPDH
dc.subjectThiols
dc.subjectTaurine Chloramine
dc.subjectInflammatory Disease
dc.subjectXanthine Oxidase
dc.subjectInflammation
dc.subjectNeutrophils
dc.subjectCardiovascular Disease
dc.subjectOxidative Stress
dc.subjectCysteine
dc.subjectGlutathione
dc.subjectEnzyme Inactivation
dc.subjectThiol Enzymes
dc.subjectAntioxidant
dc.subjectPeroxidase
dc.titleInactivation of a Thiol-Dependent Enzyme by Urate Hydroperoxide
dc.typeThesis
dc.date.updated2015-11-11T20:34:52Z
dc.language.rfc3066en
thesis.degree.disciplinePathology, Christchurch
thesis.degree.nameBachelor of Biomedical Sciences with Honours
thesis.degree.grantorUniversity of Otago
thesis.degree.levelHonours
otago.openaccessOpen
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