4.6. Enzime Guaiacol Peroxidase

The total activity of the enzyme guaiacol peroxidase (GPOX; EC 1.11.1.7) was measured according to Siegel and Galston [44] from crude protein extract. Before analysis, a composite sample of sunflower leaves was powdered in liquid nitrogen with the addition of polyvinylpolypyrrolidone (PVP) using a pestle and mortar. Crude proteins were extracted from 0.2 g of powdered tissue with 1 mL of 100 mM potassium phosphate buffer, pH 7.0, for 15 min on ice. After centrifugation for 15 min at 14,000× g and 4 °C, re-extraction with 1 mL of the same buffer was performed. The joint supernatant was used for spectrophotometrical determination of GPOX activity. The reaction mixture (pH 5.8) contained 5 mM of guaiacol, 0.2 M of KH₂PO₄, 0.2 M of Na₂HPO₄ × 12 H₂O, and 5 mM of H₂O₂. The enzymatic reaction was started by adding 40 μL of crude protein extract to the reaction mixture. The increase in absorbance was monitored at 470 nm every second for one minute and was expressed as min⁻¹ g⁻¹ of fresh mass. The protein concentration in the crude protein extract was determined according to Bradford [45] and expressed in mg g⁻¹. The specific activity of guaiacol peroxidase (GPOXs) was determined as a quotient of the total GPOX activity and protein concentration. GPOXs were expressed as a change in the absorbance at 470 nm min⁻¹ mg⁻¹ protein.