The stoichiometry of the three different types of copper in ascorbate oxidase from green zucchini squash

Deinum, J.; Reinhammar, Bengt; Marchesini, A.

doi:10.1016/0014-5793(74)80736-2

Cited by 49 publications

(23 citation statements)

References 15 publications

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“…The signal clearly demonstrates the presence of type-1 and type-2 copper as taken from the different hyperfine splittings in the gL region. The EPR spectrum reported here is practically identical to the spectrum published earlier by Nakamura et al [12] for the cucumber enzyme, and to the spectra presented by Deinum et al (2) reduced enzyme, slight excess of L-dscorbate, see Flg 5B glutamate and aspartate [13] for the enzyme from green squash. Double integration of the area under the first derivative reveals 48 f 2 % of the total chemically determined copper to be EPR detectable in frozen solution.…”

Section: Epr Spectrasupporting

confidence: 90%

“…In the case of the material investigated by Mondovi et al [8], which contained 6-8 Cu/l40000 M,, computer simulations of the EPR spectra indicated an equal amount of type-1 and type-2 copper. These results are in contrast to the ratio of 3 to 1 as found by Deinum et al [13] in a sample of ascorbate oxidase prepared by one of us (A. M.) in 1973. This preparation had 8 Cu/enzyme molecule.…”

contrasting

confidence: 93%

“…EPR spectra were simulated on a Telefunken computer TR 440 using the program developed by Dr R. Aasa, University of Goteborg, Sweden [13].…”

Section: Epr Spectroscopymentioning

confidence: 99%

“…A close examination of the EPR spectra shown by Lee and Dawson [2] clearly indicates the presence of so-called non-specific copper with large hyperfine splittings in the gll region similar to the type-2 copper in laccase or ceruloplasmin. Furthermore the absorption maximum at 880 nm [ 2 ] , which so far has not been detected in the preparations of Mondovi et al [8], Nakamura et al [12] or Deinum et al [13], and which has not been detected in laccase or ceruloplasmin either (see Table 1 in [2]), might result from another copper protein or from ascorbate oxidase with slightly modified copper sites. It should be noted that in the case of tree laccase the low-molecular-weight copper protein stellacyanin has been isolated from the same crude extract [I 81.…”

mentioning

confidence: 99%

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Ascorbate Oxidase from Cucurbita pepo medullosa

Marchesini¹,

Kroneck²

1979

European Journal of Biochemistry

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1. Ascorbate oxidase has been isolated from the green squash Cucurbitapepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20 000 M, 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 f 0.11140000 MI) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 2. The pure enzyme is characterized by the following optical purity indices: A2so/Ablo = 25 +_ 0.5, A330/A610 = 0.65 & 0.05 and A6I0/A500 = 7.0 & 0.25. The molar absorption coefficient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts to 9700 M-' em-'.3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, g , = 2.058, g, = 2.036; A , = 5.0 mT, A , = A , = 0.5 mT, for the type-2 (non-blue) copper gli = 2.242, g l = 2.053; All = 19.0 mT, A1 = 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 (%ax = 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 (l/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm ( A s = 1000 f 50 M-'

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Section: Epr Spectrasupporting

confidence: 90%

contrasting

confidence: 93%

“…EPR spectra were simulated on a Telefunken computer TR 440 using the program developed by Dr R. Aasa, University of Goteborg, Sweden [13].…”

Section: Epr Spectroscopymentioning

confidence: 99%

mentioning

confidence: 99%

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