Ascorbate Oxidase from Cucurbita pepo medullosa

Abstract

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: A₂₈₀/A₆₁₀= 25 ± 0.5, A₃₃₀/A₆₁₀= 0.65 ± 0.05 and A₆₁₀/A₅₀₀= 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.