Free radicals and oxidants are key regulators of cellular responses. Among them, hydrogen peroxide (H₂O₂) has been recognized for decades as a central mediator of redox signaling. Yet detecting it inside cells remains challenging due to its low concentration. To address this, researchers have developed genetically encoded fluorescent probes, such as those from the HyPer family, capable of showing in real time where and when H₂O₂ is produced. These probes are believed to be highly specific for hydrogen peroxide.

A new study, however, suggests that this specificity may not be so absolute. Scientists from the Research Center for Redox Processes in Biomedicine (RIDC Redoxoma) constructed a plasmid capable of promoting the efficient bacterial production of HyPer7, the newest version of these probes, and tested its reactivity under controlled laboratory conditions. The results show that, in addition to hydrogen peroxide, HyPer7 is also oxidized by peroxynitrite (ONOOH) and hypochlorous acid (HOCl), two reactive species with important biological functions.

“HyPer probes have changed the status of redox research, but it’s crucial to understand exactly what they’re detecting. Our work shows that HyPer7 isn’t as specific for hydrogen peroxide as previously thought,” says Professor Ohara Augusto from the Instituto de Química at Universidade de São Paulo (IQ-USP), who coordinated the study.

The results, published in the journal Free Radical Biology and Medicine, highlight the need for caution when interpreting experiments with HyPer7 and related probes. “The redox field is complex. You can’t perform a single experiment and draw broad conclusions. Just because you put the probe in a cell and it glowed doesn’t necessarily mean it formed hydrogen peroxide. You need to consider the context, whether there’s the possibility of forming peroxynitrite or hypochlorous acid, as happens in inflammatory conditions, for exemple. If you have any suspicions, you need to perform additional controls,” explains the researcher.

How Hyper7 Works

Hydrogen peroxide is a central metabolite in redox biology. It is generated in various cellular compartments, such as mitochondria, peroxisomes, endoplasmic reticulum, and by membrane-associated NADPH oxidases. Unlike more aggressive oxidants, H₂O₂ has limited and selective reactivity and is considered a major mediator of cellular responses.

Despite its importance, detecting H₂O₂ in real time and at precise locations remains a challenge. To address the problem, a group led by the Russian researcher Vsevolod V. Belousov developed the HyPer fluorescent probe, a protein designed to detect hydrogen peroxide in cells and organisms. Over time, this probe type was refined into Hyper7, a more sensitive version.

The strategy consists of inserting a modified fluorescent protein into the regulatory domain of OxyR, a bacterial protein with a thiol group that is highly sensitive to hydrogen peroxide. This allows HyPer7 to fluoresce in the presence of hydrogen peroxide, making its dynamics visible in living cells.

“These probes are chimeric proteins that can be precisely targeted to specific subcellular compartments. They provide a spatial location where hydrogen peroxide is being produced and where it is headed in the cell, allowing signaling to be monitored. Furthermore, they can be expressed in both cell cultures and whole organisms,” explains Augusto.

However, the researcher states that the original in vitro experiments conducted to evaluate the reaction of this probe with other biological oxidants, such as peroxynitrite and hypochlorous acid, were conducted under suboptimal conditions, which may have limited the interpretation of the results.

The Study

To conduct the study, the researchers needed considerable amounts of the protein. To guarantee its high-level bacterial expression, they constructed a novel plasmid (a small circular DNA molecule carrying additional genes) in collaboration with Luís Netto´s group from Instituto de Biociências at USP. After purifying the protein and confirming its functionality, they combined spectroscopic analysis with rapid kinetic measurements to compare how different oxidants reacted with the probe.

The experiments confirmed that H₂O₂ oxidizes HyPer7 more specifically, but showed that the probe also reacts with peroxynitrite and hypochlorous acid. The speed of these reactions varies: hydrogen peroxide oxidized the probe faster than peroxynitrite but slower than hypochlorous acid. All three reactions produced HyPer7 disulfide, although peroxynitrite and hypochlorous acid also generated additional products.

Formed by the reaction between nitric oxide and superoxide, peroxynitrite is a potent oxidant that targets thiol groups in regulatory proteins such as peroxiredoxins, key players in maintaining cellular redox balance

Hypochlorous acid, in turn, is produced by immune system cells and has even greater reactivity. It reacts with virtually all thiols available in proteins, making it a potent but less selective oxidant compared to H₂O₂ and peroxynitrite.

The results reinforce the hypothesis, already discussed by other researchers, that these probes may not detect just a specific molecule, such as hydrogen peroxide, but rather reflect the general oxidation and reduction state of thiol groups in cells. Thiol groups are sulfur-containing regions of proteins and function as sensors and regulators of redox reactions, controlling various cellular processes.

“These probes are reversible,” explains Ohara Augusto. “When hydrogen peroxide is produced, the protein is oxidized and emits fluorescence. But, like all proteins, it can also be reduced by reducing agents in the cellular environment. So, although it is normally used to detect hydrogen peroxide, it actually measures the difference in oxidation states of HyPer reactive thiols. This is the basis of redox signaling.“

Because these tools are widely used in living cells and whole organisms, demonstrating their broader reactivity has important implications for interpreting redox signaling and oxidative stress experiments.

The article “HyPer7: High-Level Bacterial Expression and Kinetics Showing Significant Oxidation by Peroxynitrite and Hypochlorous Acid,” by Edlaine Linares, Fernando R. Coelho, Natalia E. Lemos, Thiago G.P. Alegria, and Luis E.S. Netto and Ohara Augusto, can be accessed here.