4.4. Enzyme Extraction and Spectrophotometric Antioxidant Enzymatic Assays

To determine the activity of antioxidant enzymes, 0.3 g moss shoots (control and SA-treated) were ground to a fine powder in liquid nitrogen and homogenized in 2 mL of potassium phosphate extraction buffer (125 mM, pH = 7.8) using a pre-chilled mortar and pestle. The extract was centrifuged at 4 °C for 10 min at 15,000 rpm in a cooling centrifuge (HERMLE Z216 MK). The supernatant was used to determine the activity of ascorbate peroxidase (APX; EC 1.11.1.11), catalase (CAT; EC 1.11.1.6), and guaiacol peroxidase (POD; EC 1.11.1.7) according to [26] with some modifications. Molar extinction coefficient (ε) was used to calculate the enzymatic activities and expressed in terms of mmol min⁻¹ mg⁻¹ protein content. Antioxidant enzymatic activities were evaluated, and protein content was calculated after 72 h of treatment.

APX reaction mixture consisted of 125 mM potassium phosphate buffer (pH = 7.0), 5 mM Na-ascorbate, 1 mM Na₂-EDTA, 100 mM H₂O₂ and 0.1 mL plant enzyme extract was completed to a final volume 1mL at 25 °C. Enzyme activity was assayed by following the decrease in absorbance at 290 nm for 100 s (ε = 2.8 mM⁻¹ cm⁻¹).

CAT activity was determined by measuring the decrease in the H₂O₂ concentration at absorbance 240 nm for 340 s (ε = 36.6 mM⁻¹ cm⁻¹). The enzyme activity was assayed in a 1 mL reaction mixture consisting of 125 mM potassium phosphate buffer (pH = 7.0), 100 mM H₂O₂ and 0.1 mL plant enzyme extract were added to initiate the reaction.

POD activity was assayed in 1 mL reaction mixture consists of 125 mM potassium phosphate buffer (pH = 7.0), 34 mM guaiacol, 100 mM H₂O₂, 0.1 mL plant enzyme extract at 25 °C. Enzyme activity was determined by the increase in absorbance at 470 nm for 150 s (ε = 36.6 mM⁻¹ cm⁻¹). Tetra guaiacol concentration was increased in a reaction mixture because of guaiacol oxidation.