Are leaf hydrogen peroxide concentrations commonly overestimated? The potential influence of artefactual interference by tissue phenolics and ascorbate

We have examined the authenticity of two methods for determination of H2O2 in leaf tissue. We show that the high concentrations of ascorbic acid present in leaf extracts interfere with both techniques. In the chromogenic peroxidase-coupled assay, H2O2 is determined by oxidation of 3-methyl-2-benzothiazoline hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB). The method yields two phases of absorbance increase as these substrates are oxidized. We show (a) that only the first phase is dependent on extracted H2O2; (b) that the slow phase is due to phenolic-dependent generation of H2O2 during the assay; and (c) that ascorbate inhibits both phases. These effects could explain both the high values and the variable results found in the literature. The chemiluminescence method involves H2O2 enhancement of ferricyanide-induced chemiluminescence of luminol (3-amino-phthal-hydrazide). Chemiluminescence of luminol is strongly inhibited by added ascorbate, suggesting that failure to remove ascorbate from extracts will cause this method to underestimate H2O2. Using the fast phase of the peroxidase-coupled assay to estimate H2O2 in extracts from which ascorbate and phenolic compounds had been removed, we obtained leaf contents of H2O2 within the range of 40–120 nmol g–1 FW.