TEMPERATURE, PH AND UV IRRADIATION EFFECTS ON ASCORBATE OXIDASE

The activity of ascorbate oxidase (AO) purified from Cucurbita pepo medullosa was assayed after incubation at different temperatures. The optimum temperature was 35-degrees; activity was completely lost after 4 min at 65-degrees. The half-time of denaturation at 55-degrees was 21.5 min. The activation energy for the AO reaction was 10 kJmol-1. A bell-shaped pH profile of the enzymatic activity was obtained with a pH optimum of 6. The enzyme was fairly stable in the pH range 4-10. A decrease of enzymatic activity was observed upon exposure of AO to UV irradiation (t1/2 of inactivation, 8.5 min). The presence of 0.1 M mannitol made the enzyme more prone to UV damage (t1/2 of inactivation, 6.5 min). Exposure of both native and copper-free AO to UV light resulted in a decrease of intrinsic fluorescence which was larger in the presence of 0.1 M mannitol. In both cases copper-free AO was more sensitive to photoinduced damage. Under anaerobic conditions the loss of intrinsic fluorescence was lower.