Spectrofluorimetric Development and Kinetic Validation for the Quantitative Determination of Peroxidase in Pico Molar Quantities: Application in the Detection of Activity in Crude Plant Tissue

A highly sensitive catalytic spectrofluorimetric method for the determination of hydrogen peroxide and peroxidase is presented. This is based on catalytic effect of peroxidase on the oxidation of para-acetylaminophenol, a non – fluorescent compound to fluorescent probe by hydrogen peroxide in slightly basic medium and the reaction mechanism was investigated. The reaction was followed spectrofluorimetrically by measuring the fluorescence intensity of 2, 2 1 dihydroxy-5-5 1 – diacetyldiaminebiphenyl (λex = 325 nm, λem = 439 nm) at a fixed time of 5 minute from initiation of the reaction. Under the optimum conditions, peroxidase can be determined in the range 19378 pM, With a S.D. = 0.44 for 10 times measurements. The detection limit of the method was down to 0.6995 pM and LOQ value is 2.33 pM. The Michaelis – Menten constant   m K and Vmax for the reaction was found to be 103 μM and 1000 min -1 respectively. The kinetic parameters like catalytic power   H2O2 H2O2 max m K V and catalytic efficiency  eff K = 1/ slope [E]0 were found to be 9.7087 × 10 6 M -1 min -1 and 2.3620 × 10 9 M -1 min -1 respectively. Applicability of the method was tested for peroxidase activity in some vegetable plant samples.