. 2021 Aug 10;1(10):1541–1555. doi: 10.1021/jacsau.1c00288

Table 1. Summary of Reported Cellular Heme Concentrations from Known Heme Reporters or Fluorescent Heme Sensors.

reporter	heme probe	fluorescent tag(s)	K_d,heme (nM)^a^,^b	measured heme concentration (nM)	quantitation method	tested in	ref.
CYSDY-9	IsdX1, IsdC	ECFP, EYFP	63.5 ± 14.3	Cytosol 25.6 ± 5.5	FRET	HeLa	(76)
				Mitochondria 23.3 ± 4.9
				Nucleus 31.0 ± 7.0
				ER 5.4 ± 1.4^c
HS1	Cytochrome b₅₆₂	EGFP, mKATE2	3 (ferric), <1 (ferrous)	Cytosol 20–40	FRET	S. cerevisiae	(79)
				Nucleus <2.5
				Mitochondria <2.5
CHY	P. falciparum Histidine rich protein	ECFP, EYFP	∼250	Cytosol 1600	FRET	P. falciparum	(78)
apo-HRP	Horseradish peroxidase	–	–	600	peroxidase activity^d	Human lung fibroblasts (IRM90)	(73,97)
apo-HRP	Horseradish peroxidase	–	–	2100 ± 2	Reconstitution of catalytic activity^d	Human erythrocytes	(74)
apo-HRP	Horseradish peroxidase	–	270 ± 40	433 ± 125	Reconstitution of catalytic activity^d	HEK293	(75)
mAPXmEGFP^e	Ascorbate peroxidase	mEGFP	22	Cytosol 4	RET	HEK293	(77)

The heme affinity, or conversely the dissociation constant K_d,heme, is a key parameter in heme quantitation. K_d,heme values should be close to the physiological concentrations of heme (in the nM to μM range), to ensure prompt response of the sensor to changes in [heme] but also avoiding the sensor becoming either fully saturated or fully unsaturated (which is essential to study dynamic cellular processes). K_d,heme defines the fraction of total cellular heme which is available for donor–acceptor heme exchange (acceptor = apo-heme sensor). Heme sensors with the highest heme affinities will accept heme from a larger ensemble of heme donors, and vice versa. Caution is needed when comparing heme concentrations obtained with different sensors, as the sensors with lower K_d,heme will draw heme from a larger number of donors.

For ferric heme unless otherwise stated.

A relatively low concentration of heme was measured in the endoplasmic reticulum of HeLa cells compared to other cell lines (mouse melanoma B16, human HCT116 colon cancer cell, human PANC-1, hamster kidney fibroblast BHK-21, and CHO), which was interpreted as due to the high expression levels of heme oxygenase-1 in the endoplasmic reticulum surface of HeLa cells.

holo-HRP was formed by mixing apo-HRP with HEK293 lysates. The reconstitution of the catalytic activity of the HRP reporter by its binding to cellular heme was then used to measure heme concentration.

mAPX = monomeric APX; mEGFP = monomeric EGFP.