Quantifying Peroxidase Activity of PMOs

The oxygen reactivity of PMOs was measured by a time resolved quantification of H₂O₂ formation in 96-well plates (total volume of 200 μL) using a Perkin Elmer EnSpire Multimode plate reader. All reactions were performed in 100 mM sodium phosphate buffer, pH 6.0 at 22°C. Based on preliminary studies ascorbate and CDH were used in concentrations of 30 μM and 0.3 μM (0.025 mg mL^-1), respectively to prevent a limitation in the PMO reduction step. As electron donor for CDH 500 μM cellobiose was used. When ascorbate was used as reductant, it was added to a final concentration of 30 μM and enzyme assays were started by mixing 20 μL of the respective PMO with 180 μL of the ready-made assay solution containing 30 μM ascorbate, 50 μM Amplex Red and 7.14 U mL^-1 peroxidase in 96-well plate wells. In reference experiments without PMO the background signal (H₂O₂ production by CDH) was measured and subtracted from the assays. When CDH was used as reductant, the PMO assays were started by mixing 20 μL of sample solution and 20 μL CDH solution with 160 μL of the reaction mix containing cellobiose, Amplex Red and peroxidase. Initial fluorescence scans of resorufin showed highest signal intensities and lowest interference with matrix compounds when using an excitation wavelength of 569 nm and an emission wavelength of 585 nm for the selected conditions. The stoichiometry of H₂O₂ conversion to resorufin formation is 1:1. By using a high concentration of Amplex Red (50 μM) the linearity of the detection reaction was ensured and the molar ratio of Amplex Red:H₂O₂ was always greater than 50:1
[22 (link)]. The H₂O₂ concentration in the assays was far below 1 μM, which leads to a linear concentration/activity response of horseradish peroxidase, which has a K_M for H₂O₂ of 1.55 μM. The high final activity of horseradish peroxidase (7.14 U mL^-1) assures a fast conversion of the formed H₂O₂ and prevents the final reaction to be rate limiting. Additionally, it prevents the accumulation of H₂O₂, which could have detrimental effects on enzyme stability in the assay. The stability of resorufin fluorescence under these conditions was tested by addition of varying concentrations of hydrogen peroxide (0.1 – 5 μM) to the assay. A stable signal that remained constant throughout the measured period of 45 minutes was observed and maximum signal intensity was reached already during the mixing period before starting the assay. A linear relation between fluorescence and H₂O₂ concentrations in the range of 0.1 – 2 μM H₂O₂ was observed and the slope (28450 counts μmol^-1) was used for the calculation of an enzyme factor to convert the fluorimeters readout (counts min^-1), into enzyme activity. PMO activity was defined as one μmol H₂O₂ generated per minute under the defined assay conditions.