Abstract
Heme b (iron protoporphyrin IX) is an essential but potentially cytotoxic cofactor, signaling molecule, and nutritional source of iron. Its importance in cell biology and metabolism is underscored by the fact that numerous diseases, including various cancers, neurodegenerative disorders, infectious diseases, anemias, and porphyrias, are associated with the dysregulation of heme synthesis, degradation, trafficking, and/or transport. Consequently, methods to measure, image, and quantify heme in cells are required to better understand the physiology and pathophysiology of heme. Herein, we describe fluorescence-based protocols to probe heme bioavailability and trafficking dynamics using genetically encoded fluorescent heme sensors in combination with various modalities, such as confocal microscopy, flow cytometry, and microplate readers. Additionally, we describe a protocol for measuring total heme and its precursor protoporphyrin IX using a fluorometric assay that exploits porphyrin fluorescence. Together, the methods described enable the monitoring of total and bioavailable heme to study heme homeostatic mechanisms in virtually any cell type and organism.
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Acknowledgments
This work was supported by the National Institutes of Health grants GM145350 and R01NS123168, a US National Science Foundation grant 1552791, and the Vasser-Woolley endowment.
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Dominic, I.M. et al. (2024). Fluorometric Methods to Measure Bioavailable and Total Heme. In: Khalimonchuk, O. (eds) Iron Metabolism. Methods in Molecular Biology, vol 2839. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-4043-2_9
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DOI: https://doi.org/10.1007/978-1-0716-4043-2_9
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