Maurizio Tamba

Binding of copper(II) to carnosine: Raman and IR spectroscopic study

A comparative Raman and FTIR study of carnosine, a dipeptide present in several mammalian tissues, and its complexes with copper(II) at different pH values was carried out. The neutral imidazole ring gives rise to some bands that appear at different wavenumbers, depending on whether the imidazole ring is in the tautomeric form II or I. At pH 7 and 9 the molecule exists in equilibrium between the two tautomeric forms; tautomer I is predominant. Metal coordination is a factor that affects the tautomeric equilibrium, and the copper(II) coordination site can be monitored by using some Raman marker bands such as the vC(4)=C(5) band. On the basis of the vibrational results, conclusions can be drawn on the functional groups involved in the Cu(II) chelation and on the species existing in the Cu(II)-carnosine system. At neutral and basic pH the most relevant species formed when the Cu(II)/carnosine molar ratio is not very different from unity is a dimer, [Cu(2)L(2)H(-2)](0). In this complex the ligand coordinates the metal via the N (amino), O (carboxylate), and N (amide) donor atoms while the N(tau) nitrogen atoms of the imidazole rings (tautomer II) bridge the copper(II) ions. At a slightly acidic pH the two monomeric complexes [CuLH](2+) and [CuL](+) were present. In the former the imidazole ring takes part in the Cu(II) coordination in the tautomeric I form whereas in the latter it is protonated and not bound to Cu(II).

The reaction of hydrogen atoms with methionine residues : A model of reductive radical stress causing tandem protein-lipid damage

The occurrence of tandem damage, due to reductive radical stress involving proteins and lipids, is shown by using a biomimetic model. It is made of unsaturated lipid vesicle suspensions in phosphate buffer in the presence of methionine, either as a single amino acid or as part of a protein such as RNase A, which contains four methionine residues. The radical process starts with the formation of H. atoms by reaction of solvated electrons with dihydrogen phosphate anions, which selectively attack the thioether function of methionine. The modification of methionine to α‐aminobutyric acid is accompanied by the formation of thiyl radicals, which in turn cause the isomerization of the cis fatty acid residues to the trans isomers. The relationship between methionine modification and lipid damage and some details of the reductive radical stress obtained by proteomic analysis of irradiated RNase A are presented.

Kinetic studies on the formation of sulfonyl radicals and their addition to carbon-carbon multiple bonds.

The reactions of α-hydroxyl and α-alkoxyl alkyl radicals with methanesulfonyl chloride (MeSO(2)Cl) have been studied by pulse radiolysis at room temperature. The alkyl radicals were produced by ionizing radiation of N(2)O-saturated aqueous solution containing methanol, ethanol, isopropanol, or tetrahydrofuran. The transient optical absorption spectrum consisted of a broad band in the region 280-380 nm with a maximum at 320 nm typical of the MeSO(2)(•) radical. The rate constants in the interval of 1.7 × 10(7)-2.2 × 10(8) M(-1) s(-1) were assigned to an electron-transfer process that leads to MeSO(2)Cl(•-), subsequently decaying into MeSO(2)(•) radical and Cl(-). The rate constants for the addition of CH(3)SO(2)(•) to acrolein and propiolic acid were found to be 4.9 × 10(9) M(-1) s(-1) and 5.9 × 10(7) M(-1) s(-1), respectively, in aqueous solutions and reversible. The reactivity of tosyl radical (p-CH(3)C(6)H(4)SO(2)(•)) toward a series of alkenes bearing various functional groups was also determined by competition kinetics in benzene. The rate constants for the addition of tosyl radical to alkenes vary in a much narrower range than the rate constants for the reverse reaction. The stabilization of the adduct radical substantially contributes to the increase of the rate constant for the addition of tosyl radical to alkenes and, conversely, retards the β-elimination of tosyl radical.

Free radical scavenging and copper chelation: a potentially beneficial action of captopril.

Captopril (CpSH), an angiotensin converting enzyme (ACE) inhibitor, is reported to provide protection against free-radical mediated damage. The purpose of this study was to investigate, by means of pulse radiolysis technique, the behaviour of CpSH towards radiation-induced radicals in the absence and in the presence of copper(II) ions, which can play a relevant role in the metal catalysed generation of reactive oxygen species. The results indicate that the -SH group is crucial in determining the radical scavenging action of CpSH and the nature of the resulting CpSH transient products in the absence or in the presence of oxygen. In the presence of Cu(II), the -SH group is still involved in the biological action of the molecule participating both in the one-electron reduction of Cu(II) with formation of CpSSCp, and in Cu(I) chelation. This conclusion is supported by the Raman spectroscopic data which allow to identify the CpSH sites involved in the copper complex at different pH. These results suggest that CpSH may potentially inhibit oxidative damage both through free radical scavenging and metal chelation. Considering the low CpSH concentration in vivo, the metal chelation mechanism, more than the direct radical scavenging, could play the major role in moderating the toxicological effects of free radicals.

A Biomimetic Model of Tandem Radical Damage Involving Sulfur-Containing Proteins and Unsaturated Lipids

Lipidomics research, which focuses on the global changes in lipid metabolites, has recently been concerned with the type and roles of unsaturated lipids in the biological environment. The structural change induced by their conversion from the naturally occurring cis fatty acid geometry to the more thermodynamically stable trans configuration can affect membrane arrangement as well as lipid metabolism. In the biomimetic model of thiyl radical-catalyzed isomerization of cis phospholipids, it was shown that when thiyl radicals are generated in the aqueous compartment and are able to diffuse in the lipid bilayer, then the interaction with unsaturated fatty acyl chains efficiently produces trans double bonds. These findings suggested that radical-based degradation of sulfur-containing amino acid residues that are known to release diffusible thiol molecules could be the primer for tandem radical damage involving protein and lipid domains. We modeled such damage using g irradiation of lipid vesicle suspensions containing bovine pancreatic ribonuclease A (RNase A). The reaction of this protein with HC atoms was studied, and the inactivation was connected to the specific damage of sulfur moieties with release of low-molecular-weight thiols. Liposomes were prepared by using dioleoyl phosphatidyl choline (DOPC) in the form of large unilamellar vesicles (LUVET) of 100 nm diameter. The protein was added to the LUVET suspension and saturated with N2O prior to irradiation at a dose rate of 14.5 Gymin . 100 mL aliquots of the suspension were withdrawn at different irradiation times, over the interval of 2–70 min, for lipid isolation and derivatization to the corresponding fatty acid methyl esters. This was followed by GC analysis to determine the cis/trans ratio. The solid circles in Figure 1 show the percentage of trans isomers (elaidate residues) formed as a function of irradiation dose. Control experiments in the absence of RNase A or by replacing RNase A with a protein without sulfur-containing amino acids, such as histone H1 type IIA from calf thymus, did not show any isomerization. In parallel, we followed changes in the enzyme activity, as well as the transformation of the sulfur moieties by Raman spectroscopy, using lyophylized samples of aqueous solutions of native and irradiated RNase A. As expected from the radiation-induced inactivation, a residual enzymatic activity of 67% was found after exposure to only 33.3 Gy, followed by a slower decrease of the activity, which reached 50% after 500 Gy. In the Raman spectra, the S S and C S stretching bands are visible in the 420–780 cm 1 spectral range. Native RNase A has four disulfide bridges that give rise to two different disulfide bands (nS-S) at 514 and 535 cm , and four methionine residues that exhibited a band (nC-S) at 727 cm 1 (Figure 2A). Exposure of the protein at low irradiation doses (23 or 61 Gy) produced small modifications in the spectral features of the nS-S bands, as well as of the nC-S peak at 652 cm 1 that originated from cystines, as shown in Figure 2B and C. On the other hand, 23 Gy of irradiation was enough to cause significant changes in the C S bonds of methionines, as indicated by the splitting of the 727 cm 1 band into two components at 719 and 729 cm ; this suggests that a profound change in these sulfur-containing residues had taken place. The reaction sequence started from primary water radicals obtained by g radiolysis of aqueous suspensions.

A spectroscopic and pulse radiolysis study of allopurinol and its copper complex

Abstract Allopurinol (ALP), an anti-hyperuricemic drug, was investigated in the absence and in the presence of Cu(II) ions by vibrational spectroscopy and pulse radiolysis technique in order to obtain some elucidation on the mechanism of its beneficial effect against oxygen free radical-mediated damage. ALP is suggested to give protection to living organisms both by metal chelation and direct scavenging of free radicals. The Raman and IR spectra have been useful to assess the relevant interactions of ALP with Cu(II) ions, which play an important role in the metal catalysed generation of reactive oxygen species. A proposal of the complex structure was performed; the predominant species seems to be a monomeric complex where ALP interacts with the metal by one nitrogen atom of the five-membered ring and the Cue605O group. The pulse radiolysis data have given information on the behaviour of ALP towards radiation-induced radicals. ALP appears to be a good scavenger of OH, giving rise to intermediate transients, namely resonance-stabilised phenoxyl radicals. Also in the presence of Cu(II) ions, ALP reacts fast towards OH radicals.

Radical Damage Involving Sulfur-Containing Enzymes and Membrane Lipids

Free radicals induce protein modifications, often associated to many biological phenomena. This mini-review overviews the approaches we have used to elucidate the radical-induced damages on sulfur-containing enzymes, such as ribonuclease and lysozyme, and the possible post-translational mechanism of the damage to another cell compartment, such as lipid domain.

Some molecular aspects of the radiation-induced oxidative degradation of glutathione

Glutathione (GSH) is well known to have an important role as natural antioxidant. Some aspects of GSH degradation following the capture of water primary radicals in aerated or deaerated medium were investigated after γ-irradiation and by pulse radiolysis.___TAGSTART___BR___TAGEND___ Some relevant reactions involved in the GSH oxidation mechanism, leading to GSOO•-related products, were evidenced by pulse radiolysis experiments.___TAGSTART___BR___TAGEND___ From the quantitative determinations of the GSH degradation and the GSSG formation, useful indications on the fate of thiyl radicals (GS•), the most preminent sulfur centred intermediates, were drawn out in anaerobic and aerobic conditions.