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. Enzyme-based methods are appropriate for such applications. A problem commonly encountered when using enzyme-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 using the following system: horseradish peroxidase, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and a hydrogen peroxide-containing garlic extract. Included experiments evaluated the nature of assay-interference in this system, which is overwhelmingly due to reduction of generated ABTS-radicals.  Methods to circumvent the interference are presented; the methods are based on removal of interfering hydrogen donors prior to initiating the assay. The results from this work are expected to be generally applicable to other peroxidase-based assays for the quantification of hydrogen peroxide in complex biological systems.

Peroxidase-based hydrogen peroxide assays in biological systems

248th National Meeting (2014)

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