Ascorbate Oxidase from Cucurbita pepo medullosa

1 Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low‐molecular‐weight copper protein containing one type‐1 copper/20000 M r could be separated during the purification of the oxidase. The six‐step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 ± 0.1/140000 M r ) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 ± 50 units/mg protein or 1088 ± 15 units/μ copper. 2 The pure enzyme is characterized by the following optical purity indices: A 280 / A 610 = 25 ± 0.5, A 330 / A 610 = 0.65 ± 0.05 and A 610 / A 500 = 7.0 ± 0.25. The molar absorption coefficient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts to 9700 M −1 cm −1 . 3 Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type‐1 (blue) copper g z = 2.227, g y = 2.058, g x = 2.036; A z = 5.0 mT, A y = A x = 0.5 mT, for the type‐2 (non‐blue) copper g ‖= 2.242, g ⊥ , = 2.053 A ‖= 19.0mT, A ⊥ = 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type‐1 class, and one to the type‐2 class, as demonstrated by computer simulations of the EPR spectra. 4 To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L‐ascorbate or reductate must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (λ ex max = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type‐3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310nm(Δɛ= 1000 ± 50 M −1 em −1 ). Corresponding changes in EPR and fluorescence spectra have not been detected.