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Heme is an iron complex with protoporphyrin and an essential biomolecule for almost all living things, including animals, plants, and fungi. In our body, heme is involved in oxygen delivery and energy production, both of which are vital for us, and that is why we can not live without heme. The alteration in non-protein-bound heme, which is called labile heme, mediates a variety of biologically essential functions. So, it has been reported that the dysfunctions in heme homeostasis and metabolism are involved in several diseases. However, there are still very few methods to observe the alteration in heme concentration in living cells.

We have just developed a new fluorescent probe, H-FluNox, that selectively detects labile heme in living cells by expanding the Fe(II) ion-detection technology that we have developed in our laboratory. The introduction of difluoropiperidine N-oxide (Figure 1a) to rhodol fluorophore enabled the detection of labile heme with high selectivity and sensitivity. The fluorescence intensity increased up to 200 folds when H-FluNox was mixed with heme (Figure 1b). The detection limit of the probe is 100 pM, which is sensitive enough to monitor subcellular labile heme because the subcellular heme levels are maintained within the nanomolar range. The selectivity and sensitivity of H-FluNox are the best in the world so far.

Then, the probe was applied to live-cell imaging. We observed significant fluorescence signals in the cells treated with a combination of 5-aminolevulinic acid (ALA) and ferric ammonium citrate (FAC), which is known to activate heme biosynthesis. In contrast, succinylacetone (SA), a heme synthesis inhibitor, inhibited the fluorescence increase. These results indicate that the probe successfully detected labile heme in living cells. In the reported article, further applications, including nitric oxide (NO)-induced upregulation of labile heme, the potential heme-pooling ability of guanine quadruplex, heme-exporting activity of a drug-exporter protein, and heme accumulation during ferroptosis, were revealed by a series of imaging studies.

Since heme is an essential component not only for humans but also for other living organisms, our probe will contribute to the wide field of biological studies.

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Article information

Journal : Journal of the American Chemical Society
Journal title : Molecular Imaging of Labile Heme in Living Cells Using a Small Molecule Fluorescent Probe
Authors : Kanta Kawai, Tasuku Hirayama,* Haruka Imai, Takanori Murakami, Masatoshi Inden, Isao Hozumi, Hideko Nagasawa
DOI : 10.1021/jacs.1c08485

Lab HP: Laboratory of Pharmaceutical and Medicinal Chemistry https://yakka-gifu-pu.jp/