Kinetic Studies of Lactoperoxidase
|dc.contributor.advisor||Jameson, Guy N. L.|
|dc.contributor.advisor||Wilbanks, Sigurd M.|
|dc.contributor.advisor||Kettle, Anthony J.|
|dc.identifier.citation||Seidel, A. (2015). Kinetic Studies of Lactoperoxidase (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/5868||en|
|dc.description.abstract||Lactoperoxidase (LPO) is a heme-containing peroxidase present in mammalian and human exocrine secretion liquids and the mucosal surfaces of the respiratory system. It plays a key role in the innate immune defence against invading organisms, as it oxidises thiocyanate to the antimicrobial agent hypothiocyanite. Although the antioxidants urate and ascorbate are found abundantly in the lining fluids of the airway where LPO is active, their interaction with the LPO/thiocyanate system has never been studied. In this thesis, urate and ascorbate are characterised as substrates for bovine LPO. Spectroscopic studies revealed that urate and ascorbate were first oxidised by LPO via the peroxidase cycle, but under steady state conditions LPO was diverted from the classic peroxidase cycle to form compound III, depending on hydrogen peroxide concentration. Using stopped-flow spectroscopy, the rate constants for the reaction of urate with LPO compounds I and II as well as for the reaction of ascorbate with compounds I, II and III were determined. The major findings were that urate reacted fast with compound I, and that ascorbate was able to rescue compound III to re-establish the ferric form, thereby preventing enzyme inhibition. Analysis by mass spectrometry showed that urate was oxidised by LPO to produce the intermediates dehydrourate and 5-hydroxyisourate, which decayed to allantoin as final product. Hydroperoxides were shown to be formed when urate was oxidised by LPO in the presence of superoxide. Urate effectively competed with the main substrate thiocyanate for oxidation under physiological conditions, and hypothiocyanite-dependent killing of Pseudomonas aeruginosa was inhibited by urate. In vivo studies using human saliva showed that allantoin was present in saliva and its concentration was associated with LPO activity. Ex vivo studies where hydrogen peroxide was added to human saliva revealed that oxidation of urate was dependent on its concentration and LPO activity. From these findings it was concluded that urate must be a physiologically relevant substrate for LPO that is likely to impair the host defence system. It was further concluded that ascorbate may become important for the re-establishment of a functional LPO/thiocyanate defence system when local hydrogen peroxide concentrations are high. Additionally, this thesis presents an approach to replace the model of bovine LPO by human LPO. Human LPO was recombinantly produced in insect cells and purified by Strep-tag based affinity chromatography. In comparison with bovine LPO, human LPO revealed decreased activity towards the substrates tetramethylbenzidine and thiocyanate. One major difference between human and bovine LPO is the amino acid residue 254 located in close proximity to the active site. To study the role of this amino acid residue, the human LPO variant S254F was produced, where the serine residue found in human LPO is exchanged for a phenylalanine residue as found in bovine LPO. The variant showed an increased activity towards tetramethylbenzidine and a decreased Michaelis constant for thiocyanate turnover, leading to the conclusion that the amino acid residue 254 is important for substrate binding to LPO.|
|dc.publisher||University of Otago|
|dc.rights||All 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.title||Kinetic Studies of Lactoperoxidase|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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