Spectrophotometric determination of peroxidase using N, N-diethyl-p-phylenediamine sulphate and 3-Aminophenol as a chromogenic reagent: Application of the method to seeds of some fruits

A biocatalatic pathway for the determination of peroxidase using N, N‑diethyl‑p-phylenediamine sulphate (DPD) and 3-amino phenol (3-AP) is presented. The assay is based on the enzymatic consumption of hydrogen peroxide using DPD- 3AP system to give an intense blue colored compound with absorbance maxima at 660 nm. The increase in absorbance is proportional to the concentration of peroxidase in the range from 1.5-15.15 nM and 0.47 and 15.15 nM from rate and fixed time method respectively. The assay was adapted for the measurement of H2O2 at concentrations of 3.5-120 µM. The kinetic parameters like catalytic power, catalytic efficiency, catalytic constant (kcat) and specificity constant (kcat/Km) was found to be 9.78 × 10−5 µM−1 min−1, 1.483 × 10−3 min−1, 0.0245 × 103 min−1 and 0.0445 µM−1  min−1 respectively. The applicability and thermal properties of the method has been tested in different seeds of fruits extracts that showed peroxidase activity.

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