72 - A Sensitive and Cost-effective Fluorescence-based Pyrogallol red assay for H2O2: Applied to Detect H2O2 Mobility Between Culture Wells

Abstract

A novel fluorescence-based assay to quantitate hydrogen peroxide levels in biological samples was developed with the relatively inexpensive and non-toxic reagents pyrogallol red (PGR) and horseradish peroxidase (HRP). These characteristics of PGR, as well as its ability to be readily oxidized by various species and its absorbance maxima at 540 nm at physiological pH, have led to its use in a number of assays to quantitate levels of small molecule oxidants and antioxidants, metal ions, and catalase. When the reaction is catalyzed, such as by HRP, H2O2 oxidized PGR. We find that the linear range of H2O2 detection by this method is limited to the low μM range. Given its structural similarity to other anionic xanthene dyes, such as fluorescein, we hypothesized PGR would also have fluorescent properties, which might extend its linear range of H2O2 detection. We find that the reduced and oxidized forms of PGR both fluoresce, with distinct excitation and emission spectra. The linear range of H2O2 detection is significantly extended by fluorescence detection. A rapid 96-well plate assay was developed. Controls are included for 1) specifically detecting H2O2 by subtracting out any changes observed after treating samples with catalase and 2) accounting for any nucleophilic quenching of H2O2 (such as by glutathione) by pretreatment with the electrophile N-ethylmaleimide. This assay was then utilized to determine whether and to what extent H2O2 accumulated in neighboring wells in a multi-well cell culture plate, due to volatilization from a treated well. We find that H2O2 does travel from a treated well to a neighboring well.