Reactivity of hypotaurine and cysteine sulfinic acid toward carbonate radical anion and nitrogen dioxide as explored by the peroxidase activity of Cu,Zn superoxide dismutase and by pulse radiolysis.

Hypotaurine and cysteine sulfinic acid are known to be readily oxidized to the respective sulfonates, taurine and cysteic acid, by several oxidative agents that may be present in biological systems. In this work, the relevance of both the carbonate anion and nitrogen dioxide radicals in the oxidation of hypotaurine and cysteine sulfinic acid has been explored by the peroxidase activity of Cu,Zn superoxide dismutase (SOD) and by pulse radiolysis. The extent of sulfinate oxidation induced by the system SOD/H2O2 in the presence of bicarbonate (CO3(•-) generation), or nitrite ((•)NO2 generation) has been evaluated. Hypotaurine is efficiently oxidized by the carbonate radical anion generated by the peroxidase activity of Cu,Zn SOD. Pulse radiolysis studies have shown that the carbonate radical anion reacts with hypotaurine more rapidly (k = 1.1 × 10(9) M(-1)s(-1)) than nitrogen dioxide (k = 1.6 × 10(7) M(-1)s(-1)). Regarding cysteine sulfinic acid, it is less reactive with the carbonate radical anion (k = 5.5 × 10(7) M(-1)s(-1)) than hypotaurine. It has also been observed that the one-electron transfer oxidation of both sulfinates by the radicals is accompanied by the generation of transient sulfonyl radicals (RSO2(•)). Considering that the carbonate radical anion could be formed in vivo at high level from bicarbonate, this radical can be included in the oxidants capable of performing the last metabolic step of taurine biosynthesis. Moreover, the protective effect exerted by hypotaurine and cysteine sulfinate on the carbonate radical anion-mediated tyrosine dimerization indicates that both sulfinates have scavenging activity towards the carbonate radical anion. However, the formation of transient reactive intermediates during sulfinate oxidation by carbonate anion and nitrogen dioxide radical may at the same time promote oxidative reactions.

Biochemical properties of new synthetic carnosine analogues containing the residue of 2,3-diaminopropionic acid: the effect of N-acetylation.

Three novel carnosine analogues 7-9 containing the residue of L(+)2,3-diaminopropionic acid with different degree of N-acetylation instead of beta-alanine have been synthesized and characterized. Comparative analysis of hydrolysis by carnosinase revealed that the mono- and bis-acetylated compounds 8 and 9 are resistant to enzymatic hydrolysis and act as competitive inhibitors of this enzyme. The hydroxyl radical scavenging potential of the three analogues was evaluated by their ability to inhibit iron/H(2)O(2)-induced degradation of deoxyribose. The second-order rate constants of the reaction of compounds 7-9 with hydroxyl radical were almost identical to that of carnosine. These compounds were also found to act as protective agents against peroxynitrite-dependent damage as assessed by their ability to prevent nitration of free tyrosine induced by this species.

Prevention of peroxynitrite-dependent damage by carnosine and related sulphonamido pseudodipeptides.

The naturally occurring dipeptides carnosine and anserine have been proposed to act as antioxidants in vivo. We investigated whether these compounds can act as protective agents able to counteract peroxynitrite-dependent reactions. The results showed that the dipeptides efficiently protect tyrosine against nitration, alpha1-antiproteinase against inactivation and human low-density lipoprotein against modification by peroxynitrite. Carnosine exerts its protective effect at concentrations similar to those found in human tissues. In addition, some synthetic pseudodipeptides, stucturally related to carnosine but stable to hydrolytic enzymes, possess protective properties against peroxynitrite-dependent damage similar to the natural dipeptides. These pseudodipeptides may represent stable mimics of the biologically active carnosine suitable for pharmacological applications.

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 (*NO(2)) 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.

Identification of an oxidation product of aminoethylcysteine ketimine dimer.

In continuation of our previous work dedicated to the detection of the oxidation products of aminoethylcysteine ketimine dimer by oxygen reactive species, we give here data for the identification of the alpha, beta unsaturated sulfoxide as the main product of interaction of the dimer with H2O2. Identification has been done on the basis of mass spectrometry and NMR analyses of the product isolated by preparative chromatography.

Methylene blue photosensitized oxidation of cysteine sulfinic acid and other sulfinates: the involvement of singlet oxygen and the azide paradox.

The methylene blue photosensitized oxidation of cysteine sulfinic acid is investigated. Enhancement of the oxygen consumption rate in deuterium oxide suggests the involvement of singlet oxygen ((1)O(2)) in oxidation. Addition of the (1)O(2) quencher azide produced an unusual enhancement of the oxidation rate of all the sulfinates assayed. It is assumed that azide works as a one-electron carrier between (1)O(2) and the sulfur compounds. Analyses of the products indicate that the photochemical oxidation of cysteine sulfinic acid proceeds through two simultaneous mechanisms. The Type II (singlet oxygen) mechanism is responsible for oxidation of the sulfinic group to the sulfonic group with production of cysteic acid, stable to the photooxidation system, whereas the Type I (electron transfer) mechanism is involved in the degradation of cysteine sulfinic acid to acetaldehyde. Other products detected were ammonia, sulfate, and hydrogen peroxide which account for the degradation of cysteine sulfinic acid and for the excess of oxygen consumption detected during the oxidative reaction.

Hypotaurine and superoxide dismutase: protection of the enzyme against inactivation by hydrogen peroxide and peroxidation to taurine.

Hypotaurine is able to prevent the inactivation of SOD by H2O2. The protection is concentration-dependent: at 20 mM hypotaurine the inactivation of SOD is completely prevented. It is likely that hypotaurine exerts this effect by reacting with hydroxyl radicals, generated during the inactivation process, in competition with the sensitive group on the active site of the enzyme. According to this, spectral studies indicate that in presence of hypotaurine the integrity of the active site of SOD is preserved by the disruptive action of H2O2. An interesting outcome of the SOD/H2O2/hypotaurine interaction is that SOD catalyzes the peroxidation of hypotaurine to taurine. Indeed, the formation of taurine increases with the reaction time and with the enzyme concentration. Although the peroxidase activity of SOD is not specific and relatively slow compared to the dismutation of superoxide, it might represent another valuable mechanism of production of taurine.

Oxidation of hypotaurine to taurine with photochemically generated singlet oxygen: the effect of azide.

Hypotaurine is oxidized to taurine by singlet oxygen (1O2) generated with methylene blue used as a photosensitizer. The oxidation rate increases in the presence of deuterium oxide as expected for the involvement of 1O2. Addition of the 1O2 quencher azide also produced an activating effect in contrast with the expected inhibition. Azidyl radicals produced by the oxidation of azide by the horseradish peroxidase/hydrogen peroxide system stimulate the oxidation of the added hypotaurine. It is concluded that azide competes with hypotaurine for 1O2 generating the azidyl radical which is a strong one-electron oxidant transfer of the radical to hypotaurine. The hypotaurine radical is then converted into taurine, possibly through the disulfone intermediate. Formation of the sulfonic hydroperoxide is the possible intermediate in the absence of azide. The finding that the azidyl radical efficiently oxidizes hypotaurine to its metabolic product taurine raises the expectation of hypotaurine being a valuable scavenger of endogenous and exogenous radicals.

Antioxidant properties of the decarboxylated dimer of aminoethylcysteine ketimine: assessment of its ability to scavenge peroxynitrite.

The natural sulfur compound aminoethylcysteine ketimine decarboxylated dimer (AECK dimer) has been investigated for its ability to act as peroxynitrite scavenger. It has been found that the product efficiently protects against the nitration of tyrosine and the inactivation of alpha1-antiproteinase by peroxynitrite. The tyrosine nitration can be completely prevented by 100 microM AECK dimer which appears as effective as the antioxidants glutathione and N-acetylcysteine. The AECK dimer was also found to limit surface charge alteration of low density lipoprotein induced by peroxynitrite. These findings indicate that the AECK dimer is a strong protective agent against peroxynitrite damage and that it could play an important role in the defence against oxidative stress in human diseases.

Some new details of the copper-hydrogen peroxide interaction.

The addition of neocuproine (NC) or bathocuproinedisulphonate at the end of the autooxidation of Cu(I) in phosphate buffer, pH 7.4, regenerates almost entirely the O2 consumed. Other chelating agents assayed, including o-phenanthroline, cannot replace NC in promoting the O2 formation. O2 is also produced by adding NC to a mixture of Cu(II) and H2O2. Concomitant with the O2 evolution, the typical absorbance of the (NC)2Cu(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)(-)<--> Cu(I.)O2H. Addition of NC shifts the equilibrium to the right side by binding Cu(I). The released O2.- 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)2Cu(I) + O2 + 2 H+. O2 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.

An insight in the mechanism of the aminoethylcysteine ketimine autoxidation.

Oxidation of aminoethylcysteine ketimine (AECK) is followed by the change of 296nm absorbance, by the O2 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 Fe(3+) ions strongly accelerates the oxidation in the presence of EDTA or OPT. AECK reacts stoichiometrically with OPT-Fe(3+) complex producing the Fe(2+) complex which is not reoxidised by bubbling O2. 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-Fe(3+) 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 Fe(3+)) producing the radical AECK(•) as the initiator of a self propagating reaction. The radical AECK(•) reacting with O2 starts a series of reactions accounting for most of the products detected.

Antioxidant properties of the decarboxylated dimer of aminoethylcysteine ketimine.

The decarboxylated dimer of aminoethylcysteine ketimine has been investigated for a possible general protective effect against oxyradical damage. It has been found that the dimer protects brain microsomes against lipid peroxidation induced by NADPH in the presence of Fe(III)-ADP chelate or by cumene hydroperoxide. The compound also inhibits lipid peroxidation stimulated by L-dopa and related compounds in the presence of Fe(III)-ADP complex. Furthermore the dimer is able to protect deoxyribose against hydroxyl radical induced degradation. These observations suggest that the dimer is a lipid peroxidation protective agent and a free radical scavenger.

Identification of new products of S-aminoethylcysteine ketimine autoxidation.

In continuation of a previous work (Pecci et al., 1993), dedicated to the detection of the autoxidation products of S-aminoethylcysteine ketimine (AECK), we give here data for the identification of 2,3,6,7-tetrahydro-4H-[1,4]thiazino[2,3-b]thiazine, thiomorpholine-3-one and 5,5', 6,6'-tetrahydro-2,2'-dihydroxy-3,3'-bi-2H-thiazine among the products of AECK autoxidation. Identification has been done on the basis of mass spectrometry and NMR spectral analyses of the isolated products.

Aminoethylcysteine ketimine decarboxylated dimer protects submitochondrial particles from lipid peroxidation at a 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+).

Aminoethylcysteine ketimine decarboxylated dimer inhibits mitochondrial respiration by impairing electron transport at complex I level.

The product of the spontaneous dimerization and decarboxylation of aminoethylcysteine ketimine (simply named the dimer in this note) has been investigated for a possible biochemical activity. It has been found that the dimer inhibits the ADP-dependent oxidation of NAD(+)-linked substrates in rat liver mitochondria and electron transport from NADH to O2 in bovine heart submitochondrial particles (SMP). Oxidation of succinate by SMP is not impaired by concentrations of the dimer inhibiting almost totally NADH oxidation. Furthermore, the dimer did not affect the rotenone-insensitive electron transfer from NADH to menadione. These results give a preliminary indication suggesting that the dimer inhibits electron flow from NADH dehydrogenase to ubiquinone at or near the rotenone binding site(s). The dimer inhibition falls in the same range exhibited by some neurotoxins which are known to interact with the rotenone binding site.

The oxidation of aminoethylcysteine ketimine dimer by oxygen reactive species.

The prominent spontaneous reaction of aminoethylcysteine ketimine in the neutral pH range is the concentration-dependent dimerization (Hermann, 1961). The carboxylated dimer first produced loses the free carboxyl yielding the more stable decarboxylated dimer (named simply the dimer in this note). In the search for a possible biochemical activity of this uncommon tricyclic compound we have assayed whether it could interact with oxygen reactive species (H2O2, O2 (-),(•)OH) thus exhibiting a scavenging effect of possible biomedical interest. The dimer interacts with H2O2 producing compounds detectable by chromatographic procedures. The presence of Fe(2+) stimulates the oxidative reaction by yielding the hydroxyl radical (the Fenton reaction). Using the system xanthine oxidase-xanthine as superoxide producer, the dimer oxidation by O2 (-) has also been documented. Among the oxidation products the presence of taurine and cysteic acid has been established. Identification of remaining oxidation products and investigation of the possible function of the dimer as a biological scavenger of oxygen reactive species are now oncoming.

Characterization of [35S]lanthionine ketimine specific binding to bovine brain membranes.

[35S]Lanthionine ketimine binds specifically and with high affinity to bovine brain membranes. This binding has been studied in detail. It is reversible, not occurring at an uptake site or at a metabolizing enzyme and depending only weakly from ionic strength; it is affected by thiol reagents. [35S]Lanthionine ketimine specific binding is displaced only by other ketimines and by catecholamines, but not by more selective adrenergic ligands; binding parameters are reported. [3H]Adrenaline but not [3H]dihydroalprenolol is partially displaced by lanthionine ketimine. With bovine brain preparations a significant stimulation of basal adenylate cyclase activity by lanthionine ketimine is observed.

Reversible cyclization ofS-(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.