Insight 2

For plant material enzymatic assays, a Pro200 homogenizer (Pro Scientific Inc., Oxford, CT, USA) was used to prepare leaf homogenates. Three similar-sized leaves of M. giganteus were taken from each test column after 241–290 days of experiment. For determination of catalase (CAT) activity, 0.06 M sodium phosphate buffer (pH 7.4) was added to homogenates, while 0.05 M carbonate buffer (pH 10.2) was added to determine superoxide dismutase (SOD) activity. CAT activity was determined using a static method described by Góth [33 (link)], while SOD activity was measured using method described by Misra and Fridovich [34 (link)]. The homogenates were centrifuged (20 min, 4000 rpm, 4 °C) and then stored at −45 °C until the antioxidant enzyme activity assays were performed.
Measurement of protein quantity and enzymatic activity in the samples was performed using an Evolution 220 spectrophotometer (Thermo Fisher Scientific, Waltham MA, USA) and Insight 2 software (2014) (Thermo Fisher Scientific, Waltham, MA, USA). Before measuring CAT and SOD enzymatic activity, the protein content in the samples was determined using the Bradford method [35 (link)]. The protein concentration of each prepared extract was determined with reference to a standard curve, using bovine albumin as the standard protein. The detail procedure used for enzymatic analysis is described in Appendix A, Appendix A.1.4.

Optical transmittance of 30 mm-long × 20 mm-wide and 40 ± 10 µm-thick film samples was determined with a UV-Visible spectrophotometer (Thermo Scientific Evolution 220), in visible light (220–1100 nm), at room temperature. The transmittance spectra were acquired using air as blank. The spectra were analyzed by Thermo Scientific Insight 2.