Some new details of the copper-hydrogen peroxide interaction

The addition of neocuproine (NC) or bathocuproine-disulphonate at the end of the autooxidation of Cu-I in phosphate buffer, pH 7.4, regenerates almost entirely the O-2 consumed. Other chelating agents assayed, including o-phenanthroline, cannot replace NC in promoting the O-2 formation. O-2 is also produced by adding NC to a mixture of Cu-II and H2O2 Concomitant with the O-2 evolution, the typical absorbance of the (NC)(2)Cu-I complex appears to account for the complete reduction of Cu-II to Cu-I. It is concluded that the addition of H2O2 with Cu-II produces the equilibrium Cu-II(O2H)(-) (CdO2H)-O-I.. Addition of NC shifts the equilibrium to the right side by binding CuI. The released O-2(.-) then reacts with the remaining Cu-II yielding, in the presence of NC, the net reaction of 4 NC + 2 Cu-II + H2O2 --> 2 (NC)(2)Cu-I + O-2 + 2 H+. O-2 is also released in the absence of added NC provided the H2O2 concentration is increased. In these conditions the Cu-II(O2H)(-) complex undergoes other reactions leading to the copper-catalysed decomposition of H2O2. (C) 1997 Academic Press

Novel findings on the copper catalysed oxidation of cysteine

The oxidation of cysteine (RSH) has been studied by using O-2, ferricytochrome c (Cyt c) and nitro blue tetrazolium (NET) as electron accepters. The addition of 200 mu M Cu-II to a solution of 2 mM cysteine, pH 7.4, produces an absorbance with a peak at 260 nm and a shoulder at 300 nm. Generation of a cuprous bis-cysteine complex (RS-CuI-SR) is responsible for this absorbance. In the absence of O-2 the absorbance is stable for long time while in the presence of air it vanishes slowly only when the cysteine excess is consumed. The neocuproine assay and the EPR analysis show that the metal remains reduced in the course of the oxidation of cysteine returning to the oxidised form at the end of reaction when all RSH has been oxidised to RSSR. Addition of Cu-II enhances the reduction rate of Cyt c and of NET by cysteine also under anaerobiosis indicating the occurrence of a direct reduction of the acceptor by the complex. It is concluded that the cuprous bis-cysteine complex (RS-CuI-SR) is the catalytic species involved in the oxidation of cysteine. The novel finding of the stability of the complex together with the metal remaining in the reduced form during the oxidation suggest sulfur as the electron donor in the place of the metal ion

Aminoethylcysteine ketimine decarboxylated dimer protects submitochondrial particles from lipid peroxidation at concentration not inhibitory of electron transport.

In contrast with other inhibitors of the NADH dehydrogenase of the respiratory chain, the decarboxylated dimer of aminoethylcysteine ketimine protects bovine heart submitochondrial particles (SMP) from the NADH-Fe(+3)-ADP-induced lipid peroxidation. This effect, measured as inhibition of malondialdehyde formation, is concentration-dependent in the range 0.02-0.2 mM. This range of concentration is not inhibitory on NADH-oxidase activity of SMP. Furthermore the dimer is able to counteract the malondialdehyde formation stimulated by the Complex I inhibitors rotenone and N-methyl-4-phenylpyridinium (MPP+)

Reversible cyclization of S-(2-oxo-2-carboxyethyl)-L-homocysteine to cystathionine ketimine.

S-(2-oxo-2-carboxyethyl)homocysteine (OCEHC), produced by the enzymatic monodeamination of cystathionine, is known to cyclize producing the seven membered ring of cystathionine ketimine (CK) which has been recognized as a cystathionine metabolite in mammals. Studies have been undertaken in order to find the best conditions of cyclization of synthetic OCEHC to CK and for the preparation of solid CK salt product. It has been found that ring closure takes place at alkaline pH and is highly accelerated in 0.5 M phosphate buffer. The sodium salt of CK has been prepared by controlled additions of NaOH to water-ethanol solution of OCEHC under N2 atmosphere. A solid product is obtained which, dissolved in water, shows the spectral features of CK. Solutions of the sodium salt of CK show the presence of a pH depending reversible equilibrium with the open OCEHC form. Plot of the absorbance at 296 nm in function of pH indicates that at pH 9 the compound is completely cyclized while at pH 6 is totally in the open OCEHC form. At intermediate pHs variable ratios between the two forms occur. According to the results obtained by the spectral analysis, HPLC assays of the sodium salt of CK show different patterns depending on the pH of the elution buffer

Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosine

In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (O-1(2)) we found that azide, usually used as O-1(2) quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661-665]. We show here that this, effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed. (C) 2003 Elsevier Science (USA). All rights reserved

An insight in the mechanism of the aminoethylcysteine ketimine autoxidation

Oxidation of aminoethylcysteine ketimine (AECK) is followed by the change of 296 nm absorbance, by the O-2 consumption and by the HPLC analysis of the oxidation products. The oxidation is strongly inhibited by the addition of superoxide dismutase (SOD) but not by hydroxyl radical scavengers or catalase. Addition of EDTA or o-phenanthroline (OPT) favours the oxidation, probably by keeping contaminating metals in solution at the pH studied. Addition of Fe3+ ions strongly accelerates the oxidation in the presence of EDTA or OPT. AECK reacts stoichiometrically with OPT-Fe3+ complex producing the Fe2+ complex which is not reoxidised by bubbling O-2. HPLC analyses of the final oxidation products reacting with 2,4-dinitrophenylhydrazine (DNPH) confirm the AECK sulfoxide as the main product of the slow spontaneous oxidation. The detection of other oxidation products when the reaction is speeded up by the addition of the OPT-Fe3+ complex, suggests that the oxidation takes place essentially on the carbon portion of the AECK molecule in the side of the double bond. On the basis of the results presented here, a scheme of reactions is illustrated which starts with the transfer of one electron from AECK to a contaminating metal ion (possibly Fe3+) producing the radical AECK(.) as the initiator of a self propagating reaction. The radical AECK(.) reacting with O-2 starts a series of reactions accounting for most of the products detected

Formation of nitrotyrosine by methylene blue photosensitized oxidation of tyrosine in the presence of nitrite

Methylene blue photosensitized oxidation of tyrosine in the presence of nitrite produces 3-nitrotyrosine, with maximum yield at pH 6. The formation of 3-nitrotyrosine requires oxygen and increases using deuterium oxide as solvent, suggesting the involvement of singlet oxygen in the reaction. The detection of dityrosine as an additional reaction product suggests that the first step in the interaction of tyrosine with singlet oxygen generates tyrosyl radicals which can dimerize to form dityrosine or react with a nitrite-derived species to produce 3-nitrotyrosine. Although the chemical identity of the nitrating species has not been established, the possible generation of nitrogen dioxide (NO2) by indirect oxidation of nitrite by intermediately produced tyrosyl radical, via electron transfer, is proposed. One important implication of the results of this study is that the oxidation of tyrosine by singlet oxygen in the presence of nitrite may represent an alternative or additional pathway of 3-nitrotyrosine formation of potential importance in oxidative injures such as during inflammatory processes. (C) 2001 Academic Press