EuTc - Hydrogen Peroxide Quantification Agent

Europium-based dye

Product number: 02816 for 100 reactions
 

1. Background

Hydrogen peroxide (H2O2) is a reactive metabolic by-product that is a key regulator in a number of oxidative stress-related states. Functioning through NFkB and other factors, hydroperoxide-mediated pathways have been linked to asthma, arteriosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down's syndrome.

H2O2 is generated in vivo by the mitochondrial respiratory chain as well as by a range of oxidase enzymes. It is eliminated via the actions of a variety of catalases and peroxidases. Due to its implication in many disease states, there is much interest in sensitive assays for hydrogen peroxide as well as the enzymes that produce and eliminate it, and their inhibitors. This novel Europium-based dye is ideal for monitoring free and enzymatically produced H2O2, as well as the activity of catalase and peroxidase enzymes and their inhibitors. The dye makes it possible to monitor H2O2 using either fluorescent intensity or by decay time (lifetime) measurements, which help eliminate background.
 

2. Principle of Active Motif Chromeon EuTc Assays

Europium-tetracycline (EuTc) is a dye that has a number of properties that make it ideal in assays for H2O2 and peroxide-related enzymes. EuTc alone is weakly fluorescent. But EuTc combines rapidly with free H2O2 to form a EuTc-H2O2 complex that is 15-fold brighter than EuTc alone (Figure 1). Measuring fluorescent intensity makes it possible to quantify the amount of H2O2 present in a sample (Figure 2). Thus, the basic principle of the assays is based upon the following equilibrium:

EuTc (weakly fluorescent) + H2O2 ↔ EuTc-H2O2 (strongly fluorescent)



Figure 1
: Spectral properties of the EuTc dye and the EuTc-H2O2 complex.
Absorption (406 nm) and emission (617 nm) spectra
of EuTc before and after the addition of H2O2
 



Figure 2
: Calibration graph for H2O2 concentration. (F - F0) is plotted against
the concentration of H2O2, where F0 is the fluorescent intensity of EuTc before
and F is the fluorescent intensity after addition of H2O2.

Because peroxidases and catalases degrade the EuTc-H2O2 complex, EuTc can be used to measure the activity of these enzymes, which reduce the fluorescence of a sample over time or at greater enzyme concentrations (Figure 3).


Figure 3: Kinetic determination of peroxidase activity using EuTc. (A), blank;
(B) to (F), increasing amounts of peroxidase; (G), no peroxidase or H2O2 added.
 

3. Lifetime measurement to reduce background

In addition to being able to measure H2O2 levels via the increase in fluorescent intensity, EuTc makes it possible to measure the change in decay time (lifetime) of the EuTc-H2O2 complex. The use of lifetime-based measurement has significant advantages when measuring hydrogen peroxide levels in biological fluids as such samples typically have high levels of auto-fluorescence that can interfere with intensity-based measurements. Because the EuTc-H2O2 signal is relatively long lived, lifetime measurement can completely eliminate any background contributed by the light source, the biological sample (typically ~0.1-10 ns) or by free EuTc.

Dye Complex Absorption (nm) Emission (nm) Time (µs)
EuTc 406 617 30
EuTc-H2O2 406 617 60

 

4. Advantages of EuTc

Compared to conventional means of hydrogen peroxide detection and quantification (e.g. employing horse radish peroxidase as analytical enzyme) EuTc has a number of advantages regarding speed (10 min incubation), simplicity and flexibility (intensity- or lifetime-based measurement). Moreover the long decay time generally leads to an improved sensitivity. EuTc can be used in aqueous solutions of pH 7. All these properties make the EuTc dye a very interesting option for the determination of free H2O2 levels, or to assay the activity of enzymes (and enzyme inhibitors) that produce, consume or interact with free H2O2 in your biological samples.  

5. Contents & Storage

EuTc is supplied as dry powder; a sufficient amount is supplied to prepare a working solution to perform 100 reactions and is guaranteed stable for 6 months when stored at 4°C.