CAT activity was assayed in a reaction mixture containing
25 mM phosphate buffer (pH 7.0), 10 mM H2O2, and the
enzyme. The decomposition of H2O2 was followed at 240 nm
(E = 39.4 mM cm−1) [32].
The activity of guaiacol peroxidase (GPOD) was assayed following
the method described by Cakmak and Marschner [32] with
some modifications. The reaction mixture contained 25 mM phosphate
buffer (pH 7.0), 0.05% guaiacol, 10 mM H2O2, and the enzyme.
The activity was determined based on the increase in absorbance
at 470 nm caused by guaiacol oxidation (E = 26.6 mM cm−1).
DHAR (EC1.8.5.1) activity was measured by monitoring the
increase in absorbance at 265 nm (E = 14 mM−1 cm−1) caused by
the formation of AsA. The reaction mixture contained 100 mM
potassium phosphate (pH 7.0), 50 mM reduced glutathione, 5 mM
dehydroascorbate, and 100 L of the enzyme extract. The rate of
reaction was corrected for the non-enzymatic reduction of dehydroascorbate
by reduced glutathione [33].