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Glutathione and the Cytosolic Heme Pool

Student thesis: Doctoral ThesisDoctor of Philosophy

Recently glutathione (GSH) has been proposed as a key component of the cytosolic iron pool, possessing a buffering role for cytosolic iron(II), protecting it from autoxidation. However the chemical nature of the cytosolic heme pool is unknown. We have investigated whether GSH binds heme iron. If so, the resulting complex would be expected to have increased stability and solubility in aqueous solutions, compared to the extremely hydrophobic heme molecule, thereby reducing its ability to partition into membranes. An interaction between glutathione and hematin was established with the affinity constant (Ka) of glutathione for hematin determined by absorption spectroscopy to be 5x104 M-1. Using standard bioassays the influence of GSH on heme oxidase activity and the partitioning of hematin into lipid bilayers was assessed. GSH was found to stabilise hematin in the presence of H2O2 and was found to have a profound effect on the partitioning of hematin into lipid bilayers, reducing partitioning into prepared liposomes by < 70%. The presence of hematin ligated to GSH within the lysate of mammalian cells was established using synthesised [59Fe]hematin, Caco-2 cells and size exclusion HPLC. These results suggest that GSH could be the predominant ligand for the cytosolic heme pool. The effect of glutathione on absorption and catabolism of hematin in Caco-2 cells showed an initial decrease in hematin uptake and a decrease in heme oxygenase 1 expression. Hematin when ligated to GSH, in the presence of ascorbic acid and O2, was found to be rapidly degraded and whilst GSH decreases hematin partitioning into erythrocyte plasma membranes, the effect was not as dramatic as was observed in liposomes. These results did not provide further support for glutathione serving as the predominant ligand for the organic iron pool and led to the conclusion that heme is chaperoned (not by glutathione) and encapsulated within endosomes in the cytosol. It is proposed that GSH and ascorbic acid function cooperatively to rapidly ligate and degrade any heme which escapes from the endosome system into the cytosol, hence preventing ferroptosis.
Original languageEnglish
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Award date2018

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