New probes for heme detection in biological systems

  • Laura Newton

Student thesis: Doctoral ThesisPhD


Heme is an essential molecule for all aerobic life. It acts as a co-factor for a wide variety of different proteins involved in functions as diverse as oxygen transport, energy generation and catalysis. Free or unbound heme also has a less well understood role as a signalling molecule, involved in transcriptional control, protein degradation, circadian regulation and cellular redox homeostasis. However, free heme is also cytotoxic through its pro-oxidant activity and its ability to trigger inflammatory cascades. Investigations into the role of heme in physiological processes and disease have been hampered by the lack of specific heme sensors that can be used in live cells to distinguish the low concentrations of the free heme pool from the protein-bound fraction. In this thesis, potential heme sensors have been designed and synthesised based on the amino acid sequences of heme-binding regions in the proteins Bach1 and hemopexin. Synthetic peptides with a CP motif derived from Bach1 were found to bind heme in a 1:1 ratio with low micromolar affinity by UV-vis titrations. One such peptide was modified to incorporate a 7-azatryptophan residue in place of tryptophan and quenching of the red-shifted fluorescence was shown to be proportional to hemin concentration. This lead Bach1-derived peptide was used to measure heme levels in skin cell lysates and confirmed an increase in heme levels after treatment of cells with hemin or UVA irradiation. Further developments of this prototype sensor molecule have also been explored including the incorporation of a cell penetrating peptide sequence that improved uptake of the peptide by human skin cells in vitro. Conjugation of a second independent fluorophore to the heme-binding peptide was also investigated to prepare it for use in live cells, but this reduced the fluorescence of 7-azatryptophan, indicating that further optimisation of the fluorophore combination will ultimately be required. In summary, these studies provide the basis for the development of flexible peptide-based heme sensors that can be used to monitor heme levels in biological media. Further development should provide effective tools for probing the diverse physiological functions of heme, as well as aiding our understanding of how proteins such as Bach1 regulate the transcription of genes associated with heme degradation.
Date of Award10 May 2018
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorIan Eggleston (Supervisor), Rex Tyrrell (Supervisor) & Sofia Pascu (Supervisor)

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