Glutathione (GSH) is a tri-peptide (Glutamic acid, Cystein and Glycine) present in cells, showing an antioxidant effect allowing the reduction and the removal of Reactive Oxygen Species (ROS) and Peroxide from the cell. The new QuicGSHS3.0 probe from Goryo Chemicals will allow you to easily quantify intracellular concentration of GSH by Fluorescent Live cell Imaging.
Based on intramolecular FRET mechanism, the QuicGSH3.0 probes technical characteristics allow you to easily and accurately quantify intracellular glutathione (GSH):
- The dissociation constant (Kd = 3.0 mM) is optimum for measuring intracellular GSH concentrations
- Quick and reversible response to the GSH concentration changes
- Relatively slow photobleaching and low cytotoxicity enable stable measurements of intracellular GSH concentrations
Principle of Measurement and characteristics
Usually, it is not difficult to quantify an analyte in a test tube using a fluorescent probe for detection. In contrast, it is difficult to measure the intracellular concentration of the analyte, because there is usually no simple method to determine the concentration of the analyte in the environment of the cell independent of probe concentration.
Based on a intramolecular FRET mechanism (FRET = fluorescence resonance energy transfer), QuicGSH3.0 has been applied for measuring the intracellular GSH concentrations regardless of the probe concentrations.
QuicGSH3.0 (fig. 1) is a molecule formed with an orange fluorescent dye (T) and a deep-red fluorescent dye (S). At a low GSH concentration, green light excitation of T, leads to an energy transfer to S and a subsequent emission of deep-red fluorescent light. At a high GSH concentration, the energy transfer hardly occurs, so the probe emits the orange fluorescent light from the emission of T.
The mechanism enables determination of intracellular GSH concentration from a ratio of fluorescence intensities between the two wavelengths, regardless of the probe concentrations. QuicGSH3.0 has an optimum dissociation constant to GSH (Kd = 3.0 mM) for quantifying intracellular GSH concentrations which has been considered to be in a millimolar range. Because of quick and reversible response to the GSH concentration change, slow photobleaching and low cytotoxicity, QuicGSH3.0 can be used for real-time monitoring of intracellular GSH concentration changes.
Absorption and Fluorescence spectra
In the absence of GSH , the QuicGSH3.0 emission maximum
(λem) is 625 nm (fig.2) , whereas it shifts to 582 nm upon addition of GSH. Upon excitation with a green light at 520-550 nm, the ratio of fluorescence intensities
between 582 nm (Fluorescent wavelength 1) and 625 nm (Fluorescent wavelength 2) changes as the GSH concentration changes.
To determine the dissociation constant of this probe 2µM of QuicGSH3.0 (in 0.2 M phosphate buffer, pH 7,4 containing 5% DMSO) was excited at 550 nm after adding GSH of different concentrations. Fluorescent intensities were measured at 580 nm and 616 nm fig 3. Finally, the fluorescent intensity ratio (R = F580/F616) was plotted against GSH concentrations.
As mentioned above, the dissociation constant and the technical
characteristics of this probe allow a quick and reversible reaction to detect intracellular GSH. On the figure 4 you can see that the QuicGSH3.0 rapidly reacts with GSH to shift its λem from 625 to 580 nm. λem also returns quickly to 625 nm when GSH is quenched by the N-Ethylmalmeide (NEM).
The presence of GSH in cells can be a marker of different diseases. For example the lack of intracellular GSH can be connect with the progression of Parkinson Disease. It’s as also been shown that in some cancer cells (Breast, Ovarian, Lung Cancer…. *) the level of Gluthatione can be high and negatively act against cancer therapy by intercepting and inactivating ROS generated to kill the cancer cell.
Based on this observation researchers can also be interested to measure the level of GSH and ROS at the same time by fluorescent Microscopy. Goryo Chemicals are also providers of Live Cell Imaging probe for the detection of this ROS (H2O2, OH, HclO-…) you can find in our previous post or by downloading the Reactive Oxygen Species dedicated flyer.
QuicGSH3.0 is the perfect alternative to precisely and accurately quantify in Fluorescent Live Cell Imaging, intracellular GSH which can be used as a biomarker of certain diseases.
Interested by this probe? Don’t hesitate to contact your local tebu-bio office to order.
Interested by Live Cell Imaging? Browse our intereactive web page to find the Live Cell Imaging probes suited to your project needs.
- Gamcsik M. et al. 2012. Glutathione Levels in Human Tumors. Biomarkers. 2012 December ; 17(8): 671–691