Giuseppe Rotilio

An attempt to evaluate the rate of the Haber-Weiss reaction by using OH radical scavengers.

was proposed in 1934 by Haber and Weiss [l] and it was recently suggested by Beauchamp and Fridovich [2] and Peters and Foote [3] to be a source of OH’ in biological systems. This reaction would thus amplify the toxicity of superoxide, because of the high oxidizing power of ‘OH. The value of kl, which would allow one to predict the amount of ‘OH radical formed from O,, is still uncertain. Dainton and Rowbottom [4] gave a value of 3.4 M-’ s-l, while Bray [5], McClune and Fee [6] and Halliwell [7] were unable to show the occurrence of the reaction by various approaches. However a limiting value kl < 10 M-’ s-l can be deduced from their data. This value, although small in comparison to other rate constants of the 0, reactions, is still large enough for reaction (1) to be significant under some conditions. For instance at physiological pH

Competitive inhibition of Cu, Zn superoxide dismutase by monovalent anions.

Abstract Polarographic measurements showed that N3− and halides in hibit the activity of bovine Cu, Zn superoxide dismutase in a competitive fashion, as previously demonstrated for CN− and OH−. All anions increase the spin-lattice nuclear magnetic relaxation time (T1) of aqueous solutions of the enzyme as well, but the stability constants measured from T1 data are lower than those calculated from activity data. The results suggest that substrate and anionic inhibitors bind during the catalytic action at the water coordination position of the enzyme copper, and that these inhibitors may have a greater affinity for the cuprous form of the enzyme which is generated in the catalytic cycle.

Simultaneous determination of superoxide dismutase and catalase in biological materials by polarography

Abstract The polarographic method of catalytic currents applied to a wave of oxygen permits the simultaneous assay of superoxide dismutase and catalase in biological materials with high speed and reproducibility and minimal manipulation of tissues. Washed red blood cells and tissue homogenates give rise to a strong polarographic maximum, apparently due to heme proteins, which interferes with the measurement. This maximum is suppressed by addition of approximately 0.2% plasma. Therefore, the determination of the two enzymes in red blood cells can be carried out by direct addition of whole blood to the polarographic solution. Thirty microliters of blood are enough for optimal determination of both enzymes. The method can determine superoxide dismutase and catalase at concentrations as low as 2 × 10−11 m and 5 × 10−10 m , respectively, and shows a linear correlation between measured activity and enzyme levels. The average values of the two enzymes in human red blood cells was found by this method to be 2.6 × 10−6 m for catalase and 1.8 × 10−6 m for superoxide dismutase, which agree with previously reported values.

The binding of fluoride ion to bovine cuprozinc superoxide dismutase as studied by 19F magnetic relaxation

Abstract The fluoride ion has been tested as magnetic resonance probe in an aqueous solution of superoxide dimutase (SOD). While SOD EPR spectra are only slightly affected by the presence of F− up to 3 M, the nuclear 19F relaxation is very sensitive to the SOD concentration. The stability constants for the CuSOD2+-F− complex, calculated from 1 T 1 p dependence on concentration of F−, are in good agreement with those calculated from measurements in presence of competitive inhibitors (CN−, N3−) and are similar to the constants obtained from activity measurements. The plots of paramagnetic contributions to 1 T 1 and 1 T 2 against reciprocal temperaature show that the spin-spin relaxation rate is controlled by τM, which is 4 × 10−8 sec at 27°C; a ΔH‡ of ≅6 kcal mole−1 has been calculated for the exchange process. The experimental data are analyzed in terms of the Solomon-Bloembergen theory with the aim of calculating the correlation times and the Cu2+-F− bond length. The strong dependence of relaxation times on ionic strength has been interpreted according to the Bronsted-Bjerrum relationship. Finally the pH dependence on relaxation times is discussed.

Anaerobic reaction of ascorbate oxidase with ascorbate

Abstract Ascorbate oxidase is fully reduced by 4 mol of ascorbate in the absence of air, as monitored by optical and electron paramagnetic resonance spectra. At less than stoichiometric ascorbate concentration there is a slow equilibration between the 605-and 330-nm absorption bands: The 605-nm chromophore is first reduced, then its color reappears while the 330-nm absorption band decreases. Upon reoxidation with air the process takes place in the opposite direction. Intramolecular rather than intermolecular electron exchange appears to be responsible for this process. The reduced protein is about twice as fluorescent as the oxidized protein. The fluorescence quenching in the oxidized protein is related to the 330-nm absorption band rather than to the 605-nm band as previously reported for laccase.

Enzyme activity of superoxide dismutase protomers

Superoxide dismutase from eucaryotic cells are dimeric enzymes of 33 000 mol. wt. containing 1 cupric copper and 1 zinc per protomer [l] . The copper site is the active site, while the zinc has a structural role [2,3] The enzymes purified from mammalian cells are considerably resistant to denaturating agents such as 4% sodium dodecyl sulfate (SDS) and 10 M urea [3] . In particular SDS is not able to dissociate the mammalian enzymes into protomers of 16 000 mol. wt. in the absence of 2-mercaptoethanol, although it has been shown that no covalent linkages exist between the two subunits [4]. Recently it has been reported [4] that wheat germ contains two cupro-zinc isozymes, which differ from each other in so far that one of them dissociates into protomers in SDS even in the absence of 2-mercaptoethanol. As the activity of the dimer is unaffected by SDS while treatment with SDS and 2-mercaptoethanol produces inactive protomers [4] it seemed worthwhile to test the activity of the protomers obtained by the action of SDS alone. Recent studies on the mechanism of superoxide dismutase [5] suggested that only half the active sites of the bovine enzyme function in catalysis, even though the two copper centers are indistinguishable in terms of spectral features [ 51 and anion reactivity [6] This may indicate a flip-flop type of mechanism [7], in which the reaction of one copper with a superoxide ion renders the other unreactive. In the light of these considerations, we now report on the enzyme activity of wheat germ superoxide dismutase as affected by SDS and subsequent removal of the denaturating agent.

Involvement of myeloperoxidase in the metabolic activation of phagocytes: EPR studies

G. ROTILIO+, M. BRUNORI++, A. CONCETTI*, P. DRI*, and P. PATRIARCA** + Institute of Biologkai Chemistv, University of Camen’no, Camerino, and CNR Center for Molecular Biology, Rome and ++ Institute of Chemistry, Faculty of Medicine, University of Rome and CNR Center for Molecular Biology, Rome and * Laboratory of Molecular Biology, University of Camerino, Camerino and **Institute of General Pathology, University of Trieste, Trieste, Italy