Hydrogen peroxide quantification is of broad interest due to the common use of hydrogen peroxide as an oxidizing agent in industrial processing and laboratory research. Hydrogen peroxide assays are also of general importance for biological studies aimed at understanding the role of in situ generated reactive oxygen species. In the latter scenario particularly, assays amenable to high throughput processing are needed. Peroxidase-based methods are appropriate for such applications due to the high selectivity and sensitivity of enzyme catalyzed reactions. A problem commonly encountered when using peroxidase-based methods to quantify the level of hydrogen peroxide in biological samples is assay interference due the presence of assay-modifying endogenous compounds. This type of interference has limited the applicability of peroxidase/chromophore linked assays which are commonly used elsewhere for high throughput screening (e.g., the glucose oxidase/peroxidase assay for glucose quantification). Potential mechanisms of assay interference include enzyme inhibition/inactivation, substrate competition and product modification. In the present study we addressed the different mechanisms of interference, especially product (oxidized reporter molecule) modification, using the following system: horseradish peroxidase, 2,2' azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and a hydrogen peroxide-containing garlic paste extract (GPE). Methods using ABTS as an appropriate reporter molecule to circumvent the interference are based on removal
of confounding compounds, particularly referred to as natural antioxidants, prior to initiating the assay. Because confounding compounds interfere with the peroxidase-based assay by converting peroxidase-catalyzed ABTS oxidation product, ABTS•⁺ back to ABTS, prepared ABTS•⁺ was used to selectively oxidize, thus inactivate confounding compounds that would cause confounding in this assay. A calibration curve generated by using ABTS•⁺ treated GPE sample was not significantly different (p>0.05) from the curve obtained in the model buffer system. In contrast to a flat baseline generated by original GPE sample, the effectiveness of ABTS•⁺ treatment in hydrogen peroxide quantification in the presence of interference was proved. This assay allows one to simply determine the amount of hydrogen peroxide in a product in situ and thus avoids the need for sophisticated separation techniques. The limitation of the method is that the treatment required for removal of confounding compounds takes on the order of minutes and thus the method has the possibility of underestimating the hydrogen peroxide content in systems where such concentrations are changing on the seconds to minutes time scale.
The other focus of this project was a modified assay that eliminates a source of underestimation of peroxidase activity in plant extracts. Natural reducing agents endogenous to plant materials, such as phenolic compounds and ascorbic acid, may interfere with traditional peroxidase assays by reducing the oxidized product generated in the peroxidase reaction; in such assays the oxidized product is typically the reporter molecule that is monitored for enzyme quantification. The action of such reducing compounds results in an apparent lag in product development, which is interpreted as a lower enzyme activity. In such cases the time course of product production may appear sigmoidal. In some cases, these compounds may be sufficiently active as to completely obscure the rate of the reaction. This study describes a relatively simple way to alleviate complications from these compounds. The method is based on using ABTS as the reporter substrate. The oxidized product of the reaction is ABTS•⁺, which can be followed spectrophotometrically due to its relatively high molar absorptivity in the visible region. It is herein shown that one can selectively remove complicating
endogenous reducing compounds by treating the enzyme preparation with the oxidized product itself, ABTS•⁺, prior to initiating the assay. This approach is highly selective for those compounds likely to interfere with peroxidase quantification via reaction product reduction. The presented method is herein shown to remove lag phases associated with different plant extracts and, thus, more accurately reflect total peroxidase activity. The improved assay is relatively simple and should be applicable to a range of biological systems.