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The ligandin function of human GST A1-1 : thermodynamic and biophysical properties

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dc.contributor.advisor Dirr HW en
dc.contributor.advisor Vather R en
dc.contributor.author Kolobe SD en
dc.date.accessioned 2016-09-22T11:20:01Z
dc.date.available 2016-09-22T11:20:01Z
dc.date.created 2001 en
dc.date.submitted 2003 en
dc.identifier.uri http://hdl.handle.net/20.500.11892/112000
dc.description.abstract Glutathione transferases (GSTs) are detoxification enzymes which catalyze the conjugation of glutathione to toxic hydrophobic compounds, thus facilitating their elimination from the cells. These enzymes also serve as transporters of a variety of non-substrate ligands. These functions have led to the implication of GSTs in drug resistance. In an attempt to gain insight into the recognition of non-substrate ligands by GSTs, this study investigates interactions between human class Alpha GST (hGST Al-1) and bromosulfophthalein (BSP), a non-substrate ligand. The energetics of BSP binding to hGST Al-1 were studied using isothermal titration calorimetry (ITC). At 20 &#186;C hGST Al-1-BSP complex formation is characterized by a biphasic binding isotherm with K<sub>a</sub> values of 8.7 &mu;M<sup>-1</sup> and 0.1 &mu;M<sup>-1</sup> for the primary site and secondary site, respectively. The primary site binds one BSP molecule whereas the secondary site can accommodate three to four BSP molecules. Binding of BSP to both sites is enthalpically and entropically favourable, resulting in an overall favourable Gibbs free energy. The effects of ionic strength, temperature and protonation effects on the energetics of the hGST Al-1-BSP interaction were studied. The binding of BSP to the primary site displays weak ionic strength dependence, with the binding enthalpy becoming less favourable and the entropy becoming more favourable with increasing salt concentration. In contrast, binding of BSP to the secondary site is salt independent. However, the secondary site becomes prominent only in the presence of salt. The enthalpic and entropic contributions for both sites exhibited slight temperature dependence in the temperature range studied (5-25 &#186;C). Gibbs free energy showed modest changes with increasing temperature as a result of enthalpy-entropy compensation. The temperature dependence of the binding enthalpy yielded heat capacity changes of -0.34 kJ/mol/K and +0.45 kJ/mol/K for the primary and secondary sites, respectively. Calorimetric binding experiments in various buffer systems with different ionization enthalpies suggest that the binding of BSP to both sites is not coupled to a protonation/deprotonation process. The effect of BSP on hGST Al-1 CDNB-conjugation activity and ANS binding were also addressed. The binding of BSP to the primary site had a modest effect on enzyme activity and ANS binding. In contrast, the binding of BSP to the secondary site non-competitively inhibits enzyme activity and results in a competitive non-competitive inhibition with respect to ANS binding. en
dc.language English en
dc.title The ligandin function of human GST A1-1 : thermodynamic and biophysical properties en
dc.type Masters degree en
dc.description.degree MSc en

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