Hydrogen Peroxide Detection: The Methods

In at least one important way, measuring hydrogen peroxide is substantially easier than measuring superoxide. Superoxide is unstable in aqueous solution—its steady state concentration cannot be measured directly. As a result, superoxide "levels" must be determined indirectly. This can be done by either (a) measuring rates of production and removal and then correlating the two, or (b) by measuring damage to a superoxide target.

In contrast, hydrogen peroxide is relatively stable, allowing direct measurement of its concentration in many cases. The classical methods for measuring hydrogen peroxide concentrations are through direct measurement of the absorbance at 240nm of the H2O2 molecule, or through reaction of the peroxide with ferrous iron, monitored via a subsequent reaction with the dye xylenol orange. A variety of other methods are also available.

While H2O2 does not have an absorbance peak at 240nm, the absorbance at this wavelength correlates well enough with the concentration of H2O2 to allow its use as a quantitative assay, using an extinction coefficient of 43.6/Mcm (JBC 245 (9) (1970) pp2409-13). Of course, given the low extinction coefficient, this is not a very sensitive assay. In addition many cellular components will show a significant absorbance at this wavelength. However, for measuring high concentrations of H2O2, and for calibrating standard solutions, this measurement can be very useful.

A higher sensitivity, more selective assay is mediated by the oxidation of ferric iron to its ferrous state. The ferrous iron so produced will form a complex with the dye xylenol orange, resulting in a net increase in absorbance of the solution at 550nm. This assay is much more sensitive, being capable of detecting as little as 15ng H2O2 per milliliter. It is, however, subject to interferences from compounds which can oxidize ferric iron, and so requires the use of the appropriate controls as described below.

A number of other assays have been developed to measure hydrogen peroxide levels. Many of these are based on the horseradish peroxidase mediated reaction between H2O2 and some indicating reactant. For example, in the presence of H2O2 HRP will oxidize luminol, producing light. Under the proper conditions, the light output of this system can be made to be proportional to the concentration of peroxide present. This assay can be extremely sensitive, but it is also subject to many interferences, as HRP can react with a variety of different cellular substrates.

Regardless of the assay used, there are two possible sources of error must be controlled for. The first is interference from compounds other than H2O2 which are redox active and can create a signal within the assay. Such interferences can be detected by running the assay after treating the sample with catalase, which will specifically remove the H2O2. Any signal which is lost to such treatment can be confidently assigned to H2O2.

The second source of error often encountered is the scavenging of peroxide by components of the sample either before or during the assay. Cells contain elaborate defenses against peroxide damage, and such systems often continue to work during sample preparation procedures. It is therefore important to prepare samples under conditions which minimize peroxide degradation, ie short post preparation storage time, prep and storage at or below 4oC. It is also possible to “spike” a sample with a known amount of peroxide during work-up. Comparing the amount of peroxide detected in spiked vs. unspiked samples will give an indication of the extent of peroxide degradation between sampling and assay.

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