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, 21 - 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 19- 378 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 Km and Vmax for the reaction was found to be 103 µM and 1000 min-1 respectively. The kinetic parameters like catalytic power H2O2 H2O2 Vmax Km and catalytic efficiency Keff = 1/ slope [E]0 were found to be 9.7087 × 106 M -1min-1 and 2.3620 × 109 M -1 min-1 respectively. Applicability of the method was tested for peroxidase activity in some vegetable plant samples