Biochemists think hydrogen sulfide is an important gaseous biological signal. Studies have suggested that the molecule protects against oxidative stress and modulates brain activity. But there are few good ways to study the molecule in living systems. Now, researchers report a rapid, selective, and sensitive fluorescent probe that lights up on contact with hydrogen sulfide in cells (Anal. Chem. 2014, DOI: 10.1021/ac503806w).
In the past few years, the literature has swelled with reports highlighting new probes for hydrogen sulfide. However, each probe is lacking in at least one important respect, says Kyo Han Ahn of Pohang University of Science & Technology, in South Korea. For example, some probes aren’t sensitive enough to detect the small amounts of hydrogen sulfide produced by cells. Others are slow, taking more than an hour to start to glow. Cellular levels of hydrogen sulfide change quickly, Ahn says, so a probe needs to react just as rapidly to detect these fluctuations. Probes that are both fast and sensitive tend to be promiscuous as well, reacting with thiols such as homocysteine, glutathione, and cysteine, in cells. “This is the most important issue,” Ahn says. “If your probe also detects other biothiols, you cannot monitor the change of hydrogen sulfide in your system.”
To design a hydrogen sulfide probe that is fast, sensitive, and selective, Ahn’s team started with acedan, a well-known biocompatible fluorescent dye that had been used in other studies to detect thiols in cells. They modified the dye’s structure so that it was fluorescent only after hydrogen sulfide reacted with a Michael addition site on the molecule. The first version of the probe was fast and sensitive but reacted with cysteine and glutathione along with hydrogen sulfide. To increase selectivity, the researchers tweaked the probe’s structure, adding two methoxy groups to its phenyl ring. They reasoned that this extra bulk would prevent larger molecules such as cysteine and glutathione from reacting with the probe, while allowing tiny hydrogen sulfide molecules access to the reactive site.
The strategy appeared to pay off; the probe emitted a strong fluorescent signal within five minutes in a buffer containing biologically relevant concentrations of hydrogen sulfide, but it remained largely dark around cysteine, homocysteine, and glutathione. They were able to detect hydrogen sulfide concentrations as low as 50 nM. “The usual biological level of hydrogen sulfide is surely much larger than this detection limit,” Ahn says.
The researchers next incubated their probe with human cancer cells, and after 30 minutes the cells were glowing bright. Inhibitors of the hydrogen sulfide production pathway reduced the fluorescent signal by at least half. This demonstrated that the approach may work in whole organisms, Ahn says. He plans to use the probe to study how organ injury changes hydrogen sulfide signaling in animals.
“There are hundreds of fluorescent probes published, but not many are really practical for biological tests,” says Ming Xian of Washington State University. Xian hopes the team commercializes their probe so others can use it. “This looks like a really important tool that could benefit other researchers in the field,” he says.