1,721,003 research outputs found
Full, reversible copper removal from ascorbate oxidase
No full textAnaerobic treatment with cyanide of reduced ascorbate oxidase causes total depletion of copper. No significant amount of the metal is reincorporated when the apo-enzyme is incubated with cupric ions, but it is upon incubation with a stoichiometric amount (8 mol per mol of native enzyme) of a Cu(I) complex stable in air [Cu(I)(thiourea)3]Cl. The yield in reconstituted protein is higher under anaerobic conditions (85-90%) than in air (70-75%). By treatment with less than stoichiometric amounts of [Cu(I)(thiourea)3]Cl the apo-protein binds copper preferentially at the blue copper site. As a consequence the recovery of enzymatic activity is percentually lower than copper reincorporation
A ROOM-TEMPERATURE ELECTRON-PARAMAGNETIC RESONANCE STUDY OF NATIVE AND FLUORIDE-REACTED VIETNAMESE AND JAPANESE LACQUER-TREE LACCASES - DIFFERENCES FROM LIQUID-NITROGEN SPECTRA
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Unmediated heterogeneous electron transfer reaction of ascorbate oxidase and laccase at a gold electrode
No full textThe unmediated electrochemistry of two large Cu-containing proteins, ascorbate oxidase and laccase, was investigated by direct-current cyclic voltammetry. Rapid heterogeneous electron transfer was achieved in the absence of promoters or mediators by trapping a small amount of protein within a solid, electrochemically inert, tributylmethyl phosphonium chloride membrane coating a gold electrode. The problems typical of proteins in solution, such as adsorption on the electrode surface, were avoided by this procedure. In anaerobic conditions, the cyclic voltammograms, run at a scan rate of up to 200 mV/s, showed the electron transfer process to be quasi-reversible and diffusion-controlled. The pH-dependent redox potentials (+/-360 mV and +400 mV against a normal hydrogen electrode at pH 7.0 for ascorbate oxidase and laccase respectively and +390 mV and +410 mV at pH 5.5) were similar to those of the free proteins. The same electrochemical behaviour was recorded for the type 2 Cu-depleted derivatives, which contain reduced type 3 Cu, whereas the apoproteins were electrochemically inactive. Under aerobic conditions the catalytic current intensity of holoprotein voltammograms increased up to approx. 2-fold at a low scanning rate, with unchanged redox potentials. The voltammograms of type 2 Cu-depleted proteins and of apoproteins were unaffected by the presence of oxygen. This suggests that electron uptake at the electrode surface involves type 1 Cu and that only in the presence of oxygen is the intramolecular electron transfer to other protein sites rapid enough to be observed. The analogy with available kinetic results is discussed
The role of copper in the stability of ascorbate oxidase towards denaturing agents.
No full textThe susceptibility of native, type-2 Cu-depleted and fully Cu-depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation. The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography
Reassessment of copper stoichiometry in ascorbate oxidase
No full textA very pure ascorbate oxidase solution was obtained by dissolving a crystalline sample of the enzyme. The ratio between 280 and 610 nm absorbancies was 22.5. It contained 8.0 ± 0.2 Cu ions, 50% EPR detectable, per dimeric molecule (140,000 M.W.) with a molar extinction coefficient of 10,000 cm-1 at 610 nm. Two Cu ions were removed by treatment with N,N-diethyldithiocarbamate. The optical blue absorption band was unaffected, while two EPR detectable Cu ions were lost, with disappearance of the type 2 Cu signal. It is concluded that native ascorbate oxidase contains two type 1, two type 1, two type 2, and four type 3 Cu ions. © 1988 Academic Press, Inc
On the oxidation and reduction reactions of bovine serum amine oxidase: a kinetic study.
No full textThe presteady-state and steady-state kinetics of bovine serum amine oxidase (BSAO) were analyzed by stopped-flow transient spectroscopy. A simplified model of the catalytic cycle was found to describe the experimental data and the rate constants of the individual steps were used to calculate Michaelis parameters that agree with the direct determinations. In spite of many studies on selected reactions from the catalytic cycle, this is amongst the first efforts to provide a comprehensive kinetic description of the reactions of BSAO, whose results can be compared with the steady-state parameters. The reoxidation reaction by dioxygen is more complex than previously thought, in agreement with a recent report [Su, Q. & Klinman, J.P. (1998) Biochemistry 37, 12513-12525], and occurs in at least two steps whose rate constants, previously undetermined, have been measured. The reaction of the oxidized enzyme with the amine substrate is poorly determined in this type of experiment, thus irreversible combination with aromatic hydrazine inhibitors was used as a model system, demonstrating that the mechanism and rate constants of their reaction is fully compatible with an accurate description of the catalytic cycle with the physiological substrate. These results constitute a simplified, yet complete and consistent, description of the catalytic cycle and offer an interesting comparison with those obtained on plant amine oxidases; two steps of the catalytic cycle are significantly slower in BSAO than in pea seedling or lentil seedling amine oxidases, namely the reoxidation and the trans-iminative proton abstraction occurring in the enzyme-substrate complex. The former difference is rationalized as being due to the low to zero concentration of the semiquinolamine-radical intermediate, while the latter is less easily interpreted
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