The following analytical methods (Green and Hill 1984; peroxidase activity determination from SIGMA) were used to detect changes in hydrogen peroxide concentration changes.
Reduction of H2O2 catalyzed by peroxidase in the presence of pyrogallol: a mixture of pyrogallol (at a concentration not lower than the H2O2 one) and peroxidase (POD) was added to the H2O2 aqueous solution. The measured change in absorbance at 420 nm corresponding to purpurogallin formation (e = 248/M/cm) is directly related to the H2O2 concentration decrease according to Eqs. 4 and 5:
The co-oxidation of phenol and antipyrin was catalyzed by peroxidase. A mixture of phenol, 4-amino-antipyrin (at concentrations not lower than half the concentration of H2O2) and peroxidase was added to the H2O2 aqueous solution. The change in absorbance was recorded at 505 nm (e = 6.4 X 103/M/cm), which corresponds to the wavelength maximum of the absorption band of the quinone-imine produced by the co-oxidation of phenol and antipyrin catalyzed by the peroxidase.
For high concentrations of H2O2 (» 10-2M), a direct spectroscopic measurement was used. The optical density of the solution was recorded at 240 nm and the resulting concentration was calculated using a value of 39.4/mol/cm (Aebi 1984) for the molar extinction coefficient of H2O2.
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