A quick and sensitive method for the quantification of peroxidase activity of organic surface soil from forests

The purpose of the present study was to test the non-mutagenic compound 3,3′,5,5′-tetramethylbenzidine (TMB) as a model substrate for peroxidase in forest topsoil, as an alternative to the conventional substrate l-3,4-dihydroxyphenylalanine (l-DOPA). TMB was highly sensitive; linear absorbance changes of 0.6 were achieved within 20 min for 1000-fold diluted soil. Brief heating (denaturation) of the soil suspension gave a 34-fold reduction of TMB oxidation, indicating that the reaction measured by TMB was indeed an enzymatic reaction. TMB oxidation showed a narrow peak at pH 4.4. A proportional decrease in peroxidase activity, when the soil suspension was diluted, demonstrated that TMB estimates of peroxidase activity are directly comparable when corrected for differences in sample size. Oxidation of TMB was slow in the absence of H2O2 suggesting that TMB is a poor substrate for phenol oxidases. TMB oxidation was tested in nine different forest topsoils. The peroxidase activity, when normalised to the amount of soil organic matter, ranged from 1.4±0.1 Δabs450 h−1 mg−1 to 34.9±4.3 Δabs450 h−1 mg−1. In comparison, l-DOPA oxidation by soil peroxidases and commercial peroxidases gave inconsistent results, suggesting that one should be cautious when using l-DOPA as a soil peroxidase substrate. The high sensitivity of TMB, compared to l-DOPA, and the low interference from phenol oxidase and humic substances suggest that TMB is a better substrate than l-DOPA for estimation of peroxidase activity of forest topsoil.