Determination of kinetic parameters of the enzymatic reaction between soybean peroxidase and natural antioxidants using chemometric tools

The oxidation of eugenol, isoeugenol and vanillin natural antioxidants catalyzed by the soybean peroxidase enzyme was studied using uv-vis spectroscopy. An experimental design was used to optimize the different variables. The multivariate curve resolution method was used to obtain the profiles of antioxidant absorbance's as a function of time due to uv-vis absorption bands of both antioxidants and the enzymatic reaction product/s show a strong overlap. From these results, apparent Michaelis-Menten constants as well as the kinetic parameters k1 and k3 involved in the catalytic cycle of peroxidases were calculated. The antioxidant apparent acidity constants were also determined at different pH's from uv-vis spectrophotometric measurements. Values of k1 were (0.6 ± 0.1) × 105 M−1 s−1, (2.0 ± 0.2) × 105 M−1 s−1 and (7.0 ± 0.5) × 106 M−1 s−1 and k3 (4.0 ± 0.2) × 105 M−1 s−1, (6.0 ± 0.6) × 105 M−1 s−1 and (6.0 ± 0.9) × 106 M−1 s−1 for eugenol, isoeugenol and vanillin, respectively.