2.2. Peroxidase Activity Assay

Peroxidase activity was measured by the method previously described [16] with some modifications. The method is based on the generation of tetraguaiacol from guaiacol by peroxidase in the presence of H₂O₂. Measurements were performed by mixing 1 ml of substrate solution containing 4 mM guaiacol, 2 mM H₂O₂, and 50 mM sodium phosphate buffer, with pH 7.0 as substrate buffer with 10 µl of Tg-Hb at 3 mg/L in substrate buffer. After brief shaking to mix thoroughly, the mixture was measured continuously for 2 min at room temperature for the absorbance at 470 nm using a Shimadzu UV-1800 spectrophotometer connected with a recorder. As the reaction consists of the formation of one molecule of tetraguaiacol and 4 molecules of H₂O from 4 molecules of guaiacol and 2 molecules of H₂O₂, the enzyme activity was calculated from ΔA₄₇₀/min according to the following formula: ε(tetraguaiacol)₄₇₀ = 26600 M⁻¹cm⁻¹.