Structural, functional and stability characterisation of human glutathione S-transferase Pi

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dc.contributor.authorMhlanga, Donald
dc.date.accessioned2019-05-24T11:05:54Z
dc.date.available2019-05-24T11:05:54Z
dc.date.issued2018
dc.descriptionA dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements in fulfilment of the degree for Master of Science. October 2018en_ZA
dc.description.abstractGlutathione S-transferases (GSTs) are Phase II detoxification enzymes that catalyse the conjugation of glutathione (GSH) to non-polar xenobiotic compounds to form water-soluble metabolites. Despite the low level of sequence similarity, the different GST classes follow the same canonical fold. hGSTP1-1 belongs to the Pi class and is involved in detoxification, as well as other non-classical roles such as regulating the MAP kinase pathway, protecting cells from nitrosative stress and regulating the function of 1-Cys peroxiredoxin. The structure, function and stability of GSTP1-1 was characterised to gain a better understanding of the general characteristics of the enzyme. The heterologous expression of hGSTP1-1 in Escherichia coli produces high yields of the enzyme that is then purified using immobilised metal affinity chromatography. A GSH-CDNB conjugation assay shows that the enzyme catalyses this reaction with a specific activity of 55.5 μmol/min/mg. The enzyme also binds 8-anilinonaphthalene-1-sulfonic acid (ANS), resulting in a blue shift and a two-fold increase in the fluorescence intensity of ANS. Far-UV circular dichroism shows that hGSTP1-1 is a predominantly alpha-helical protein, while intrinsic fluorescence studies show that the enzyme has Trp residues. Studies done using size exclusion HPLC show that the protein adopts a monomeric structure when exposed to high salt concentrations. Thermal unfolding of hGSTP1-1 shows that the enzyme unfolds irreversibly when exposed to increasing temperatures. Urea denaturation of the enzyme follows a two-state model (N2 ↔ 2U) and shows that domain 1 and domain 2 unfold in a cooperative manner.en_ZA
dc.description.librarianE.R. 2019en_ZA
dc.format.extentOnline resource (69 leaves)
dc.identifier.citationMhlanga, Donald, (2018) Structural, functional and stability characterisation of human glutathione S-transferase Pi, University of the Witwatersrand, Johannesburg, https://hdl.handle.net/10539/27229.
dc.identifier.urihttps://hdl.handle.net/10539/27229
dc.language.isoenen_ZA
dc.subject.lcshGlutathione transferase
dc.subject.lcshIsoleucine
dc.titleStructural, functional and stability characterisation of human glutathione S-transferase Pien_ZA
dc.typeThesisen_ZA
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