Assay of Guaiacol Peroxidase Activity

Guaiacol peroxidase (GPX; EC 1.11.17) activity was measured spectrophotometrically at 25°C using the method previously described by Tatiana et al. (1999). The reaction mixture (2.0 ml) consisted of 0.05 M potassium phosphate buffer (pH 7.0), 2 mM H₂O₂, and 2.7 mM guaiacol. The reaction was initiated by the addition of 0.1 ml of the enzyme extract. The initial rate of guaiacol oxidation was measured by the formation rate of tetra guaiacol and was measured at 470 nm (Δε = 26.6 mM/cm). One unit was defined as the amount of enzyme required to catalyze one micromole of hydrogen peroxide conversion, with guaiacol as hydrogen donor, per minute under specified conditions.