Fluorescent probe to detect intracellular labile heme HemeGREEN™＜Intracellular Labile Heme Detection Reagent＞

HemeGREEN™ is a fluorescent probe that enables visualization of intracellular labile heme in living cells. It exhibits high selectivity for labile heme over other reactive species such as ferrous iron (Fe²⁺) and heme tightly bound to proteins.

• Intracellular Labile Heme Analysis
• Principle
• Features
• Reference Data
• Assay Overview
• Example Data
• Original article
• Product information

Intracellular Labile Heme Analysis

Heme is a coordination compound of protoporphyrin IX and an iron ion at its center. In bioligical systems, it plays a crucial role as a cofactor of heme proteins, such as oxygen-transport/storage proteins like hemoglobin and myoglobin, and metabolic or redox proteins like peroxidases and cytochrome P450. While most cellular heme exists tightly bound within heme proteins, a small amount of labile (unbound) heme also exists and has recently attracted attention. Several heme-responsive transcription factors, such as Bach2, have been identified, implicating labile heme in cellular differentiation, circadian rhythm, and disease pathogenesis. Due to its high lipophilicity and redox reactivity compared to free iron ions, labile heme is also thought to contribute to oxidative damage of biomolecules. However, analytical tools for detecting and visualizing intracellular labile heme have been limited, and its dynamics remain poorly understood.

HemeGREEN™ is a small molecule fluorescent probe to enable selective detection of intracellular labile heme.

HemeGREEN™ consists of a Rhodol fluorophore with acetyl groups for enhancing cell permeability and a heme-responsive moiety. It exhibits almost no fluorescence in its native form. Once inside the cell, the acetyl groups are cleaved by intracellular esterases to yield deacetylated HemeGREEN™, which still does not fluoresce unless labile heme is present. In the presence of labile heme, the probe reacts with the heme to emit green fluorescence with a maximum emission at 535 nm.
※ HemeGREEN™ is designed exclusively for live-cell imaging and is not suitable for in vitro assays.

※ Optimize probe concentration and incubation time depending on the cell type.
※ Fluorescence dye may diffuse over time; imaging is recommended immediately after washing.

HeLa cells were treated overnight with 1 mM 5-aminolevulinic acid (ALA) and 10 μM ferric ammonium citrate (FAC) to promote heme biosynthesis. In parallel, cells were co-treated with 1 mM succinylacetone (SA), an inhibitor of ALA dehydratase, and 1 μM ZnPPIX, an inhibitor of heme oxygenase-1. The following day, cells were stained with HemeGREEN™ for 30 minutes and observed by confocal microscopy. ALA and FAC increased fluorescence, indicating enhanced labile heme levels. This signal was suppressed by SA, suggesting inhibition of heme biosynthesis. Co-treatment with ZnPPIX further enhanced fluorescence, indicating inhibition of heme degradation and accumulation of labile heme.

It has been known that NO-treatment of heme-binding proteins causes transient release of heme from the proteins. HemeGREEN™ detected this transient change of labile heme in living cells. HeLa cells were incubated with the NO donor NOC-5 for 20 minutes, followed by staining with HemeGREEN™ for 30 minutes. Confocal microscopy revealed a concentration-dependent increase in fluorescence, indicating that NO increased intracellular labile heme levels.