Cary 300 bio uv vis

Chlorophyll was assayed according to Arnon (1949) (link) and calculated as described by Porra (2002) (link). Pulverized leaves (about 2 mg) or isolated chloroplasts (about 0.5 mg) were extracted in 1 ml of acetone:H₂O (4:1 v/v) with gentle agitation in the dark for 24 h at 4 °C. The extracts were centrifuged, and absorbance was measured using a spectrophotometer (Cary 300 Bio UV/VIS, Varian, Middelburg, Netherlands). Protein content was determined by the Bradford method with bovine serum albumin as a standard (Bradford, 1976 (link)).

Rubisco activity was assessed according to Parry et al. (2002) (link), with a few modifications, by coupling its activity to NADH oxidation using phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. Leaf samples (500 mg) were homogenized with 1 ml extraction buffer (0.1 M Tris pH 7.8, 5 mM MgCl₂, 5 mM DTT, 0.1 mM EDTA, 1.5% polyvinylpyrrolidone) and centrifuged at 16 000 g for 10 min at 4 °C. The supernatant was used for determining the initial and total activity of Rubisco. The oxidation of NADH was measured at 340 nm using a spectrophotometer (Cary 300 Bio UV/VIS, Varian, Middelburg, Netherlands). Activity was calculated using a molar extinction coefficient of 6230 M^–1 cm^–1.

Tissue weighing 200 mg was homogenized in 1 ml of 0.1 M phosphate buffer at pH 6.5. The supernatant was used to determine the activity of enzymes according to Urbanek et al. (1991) and protein content as above (Bradford, 1976 (link)). The reaction mixture for catalase consisted of 100 µl extract in 3 ml phosphate buffer at pH 6.8 (Urbanek et al., 1991 ). The detoxification of H₂O₂ was measured at 240 nm (Cary 300 Bio UV/VIS, Varian, Middelburg, Netherland), with ε=43.6 M cm⁻¹. SOD activity was quantified according to Sen Gupta et al. (1993) (link) and peroxidase according to Malik and Singh (1980) , with ε=25 mM⁻¹ cm⁻¹. APX activity was assessed according to Yoshimura et al. (2000) (link) by monitoring the rate of ascorbate oxidation at 290 nm, with ε=2.8 mM⁻¹ cm⁻¹. GR activity was measured by following the increase in absorbance in the presence of GSSG and DTNB (Sairam et al., 2002 ), with ε=13.6 mM⁻¹ cm⁻¹.