Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom.

The mechanism of DNA damage by hydrazine in the presence of metal ions was investigated by DNA sequencing technique and ESR-spin trapping method. Hydrazine caused DNA damage in the presence of Mn(III), Mn(II), Cu(II), Co(II), and Fe(III). The order of inducing effect on hydrazine-dependent DNA damage (Mn(III) greater than Mn(II) approximately Cu(II) much greater than Co(II) approximately Fe(III)) was related to that of the accelerating effect on the O2 consumption rate of hydrazine autoxidation. DNA damage by hydrazine plus Mn(II) or Mn(III) was inhibited by hydroxyl radical scavengers and superoxide dismutase, but not by catalase. On the other hand, bathocuproine and catalase completely inhibited DNA damage by hydrazine plus Cu(II), whereas hydroxyl radical scavengers and superoxide dismutase did not. Hydrazine plus Mn(II) or Mn(III) caused cleavage at every nucleotide with a little weaker cleavage at adenine residues, whereas hydrazine plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the GTC sequence. ESR-spin trapping experiments showed that hydroxyl radical is generated during the Mn(III)-catalyzed autoxidation of hydrazine, whereas hydrogen atom adducts of spin trapping reagents are generated during Cu(II)-catalyzed autoxidation. The results suggest that hydrazine plus Mn(II) or Mn(III) generate hydroxyl free radical not via H2O2 and that this hydroxyl free radical causes DNA damage. A possibility that the hydrogen atom releasing compound participates in hydrazine plus Cu(II)-induced DNA damage is discussed.     

Iron(III)- and copper(II) complexes of an asymmetric, pentadentate salen-like ligand bearing a pendant carboxylate group

The equilibrium and solution structural properties of the iron(III) and copper(II) complexes of an asymmetric salen-like ligand (N,N'-bis(2-hydroxybenzyl)-2,3-diamino-propionic acid, H(3)bhbdpa) bearing a pendant carboxylate group were characterized in aqueous solution by potentiometric, pH-dependent electron paramagnetic resonance (EPR) and UV-Vis (UV-Visible) measurements. In the equimolar systems the pentadentate ligand forms very stable, differently protonated mononuclear complexes with both metal ions. In the presence of iron(III) {NH, PhO(-), COO(-)}, {2NH, 2PhO(-), COO(-)} and {2NH, 2PhO(-), COO(-), OH(-)} coordinated complexes are dominant. The EPR titrations reflected the presence of microscopic complex formation pathways, leading to the formation of binding isomers in case of Cu(H(2)bhbdpa)(+), Cu(Hbhbdpa) and Cu(bhbdpa)(-). The {2NH, 2PhO(-)+COO(-)/H(2)O} coordinated Cu(bhbdpa) is the only species between pH 6-11. At twofold excess of metal ion dinuclear complexes were detected with both iron(III) and copper(II). In presence of iron(III) a mu-carboxylato-mu-hydroxo-bridged dinuclear complex (Fe(2)(bhbdpa)(OH)(3)) is formed from Fe(H(2)bhbdpa)(2+) through overlapping proton release processes, providing one of the rare examples for the stabilization of an endogenous carboxylate bridged diiron core in aqueous solution. The complex Cu(2)(bhbdpa)(+) detected in the presence of copper(II) is a paramagnetic (S=1) species with relatively weakly coupled metal ions.     

Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes

Redox-active metal ions such as Fe(II)\(III) and Cu(I)\(II) have been proposed to activate reactive oxygen and nitrogen species (RONS) and thus, perpetuate oxidative damage. Here, we show that concentrations of metal ions and EDTA complexes with superoxide-destroying activities equivalent to 1 U SOD are Fe(III) 5.1 microM, Mn(II) 0.77 microM, Cu(II)-EDTA 3.55 microM, Fe(III)-EDTA 2.34 microM, and Mn(II)-EDTA 1.38 microM. The most active being the aquated Cu(II) species which exhibited superoxide-destroying activity equivalent to 2U of SOD at 0.29 microM. Hydrogen peroxide-destroying activities were as follows Fe(III)-EDTA ca. 70 U/mg and aquated Fe(III) 141 U/mg. In contrast, DTPA prevented superoxide-destroying activity and significantly depleted hydrogen peroxide-destroying activity. In conclusion, non-protein bound transition metal ions may have significant anti-oxidant effects in biological systems. Caution should be employed in bioassays when chelating metal ions. Our results demonstrate that DTPA is preferential to EDTA for inactivating redox-active metal ions in bioassays.     

Mechanism of site-specific DNA damage induced by methylhydrazines in the presence of copper(II) or manganese(III).

DNA damage induced by methylhydrazines (monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine) in the presence of metal ions was investigated by a DNA sequencing technique. 1,2-Dimethylhydrazine plus Mn(III) caused DNA cleavage at every nucleotide without marked site specificity. ESR-spin-trapping experiments showed that the hydroxyl free radical (.OH) is generated during the Mn(III)-catalyzed autoxidation of 1,2-dimethylhydrazine. DNA damage and .OH generation were inhibited by .OH scavengers and superoxide dismutase, but not by catalase. The results suggest that 1,2-dimethylhydrazine plus Mn(III) generates .OH, not via H2O2, and that .OH causes DNA damage. In the presence of Cu(II), DNA cleavage was caused by the three methylhydrazines frequently at thymine residues, especially of the GTC sequence. The order of Cu(II)-mediated DNA damage (1,2-dimethylhydrazine greater than monomethylhydrazine approximately 1,1-dimethylhydrazine) was not correlated with the order of methyl free radical (.CH3) generation during Cu(II)-catalyzed autoxidation (monomethylhydrazine greater than 1,1-dimethylhydrazine much greater than 1,2-dimethylhydrazine). Catalase and bathocuproine, a Cu(I)-specific chelating agent, inhibited DNA damage while catalase did not inhibit the .CH3 generation. The order of DNA damage was correlated with the order of ratio of H2O2 production to O2 consumption observed during Cu(II)-catalyzed autoxidation of methylhydrazines. These results suggest that the Cu(I)-peroxide complex rather than the .CH3 plays a more important role in methylhydrazine plus Cu(II)-induced DNA damage.     

Complexation of desferricoprogen with trivalent Fe, Al, Ga, In and divalent Fe, Ni, Cu, Zn metal ions: effects of the linking chain structure on the metal binding ability of hydroxamate based siderophores

Complexes of the natural siderophore, desferricoprogen (DFC), with several trivalent and divalent metal ions in aqueous solution were studied by pH-potentiometry, UV-Vis spectrophotometry and cyclic voltammetry. DFC was found to be an effective metal binding ligand, which, in addition to Fe(III), forms complexes of high stability with Ga(III), Al(III), In(III), Cu(II), Ni(II) and Zn(II). Fe(II), however, is oxidized by DFC under anaerobic conditions and Fe(III) complexes are formed. By comparing the results with those of desferrioxamine B (DFB), it can be concluded that the conjugated beta-double bond slightly increases the stability of the hydroxamate chelates, consequently increases the stability of mono-chelated complexes of DFC. Any steric effect by the connecting chains arises only in the bis- and tris-chelated complexes. With metal ions possessing a relatively big ionic radius (Cu(II), Ni(II), Zn(II), In(III)) DFC, containing a bit longer chains than DFB, forms slightly more stable complexes. With smaller metal ions the trend is the opposite. Also a notable difference is that stable trinuclear complex, [Cu(3)L(2)], is formed with DFC but not with DFB. Possible bio-relevance of the Fe(II)/Fe(III) results is also discussed in the paper.     

Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.

Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid A beta peptides has been linked to peroxide generation in cell cultures by an unknown mechanism. We now show that human A beta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II), setting up conditions for Fenton-type chemistry. Spectrophotometric experiments establish that the A beta peptide reduces Fe(III) and Cu(II) to Fe(II) and Cu(I), respectively. Spectrochemical techniques are used to show that molecular oxygen is then trapped by A beta and reduced to H2O2 in a reaction that is driven by substoichiometric amounts of Fe(II) or Cu(I). In the presence of Cu(II) or Fe(III), A beta produces a positive thiobarbituric-reactive substance (TBARS) assay, compatible with the generation of the hydroxyl radical (OH.). The amounts of both reduced metal and TBARS reactivity are greatest when generated by A beta 1-42 > A beta 1-40 > rat A beta 1-40, a chemical relationship that correlates with the participation of the native peptides in amyloid pathology. These findings indicate that the accumulation of A beta could be a direct source of oxidative stress in Alzheimer's disease.     

Synthesis, characterization and antitumor studies of Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) complexes of N-salicyloyl-N'-o-hydroxythiobenzhydrazide

A new ligand N-salicyloyl-N'-o-hydroxythiobenzhydrazide (H2Sotbh) forms complexes [Mn(HSotbh)2], [Fe(Sotbh-H)(H2O)2], [M(Sotbh)] [M=Co(II), Cu(II) and Zn(II)] and [Ni(Sotbh)(H(2)O)2], which were characterized by various physico-chemical techniques. M?ssbauer spectrum of [Fe(Sotbh-H)(H2O)2] reveals the quantum admixture of 5/2 and 3/2 spin-states. Mn(II), Cu(II) and Ni(II) complexes were observed to inhibit the growth of tumor in vitro, whereas, Fe(III), Co(II), Zn(II) complexes did not. In vivo administration of Mn(II), Cu(II) and Ni(II) resulted in prolongation of survival of tumor bearing mice. Tumor bearing mice administered with Mn(II), Cu(II) and Ni(II) complexes showed reversal of tumor growth associated induction of apoptosis in lymphocytes. The paper discusses the possible mechanisms and therapeutic implication of the H2Sotbh and its metal complexes in tumor regression and tumor growth associated immunosuppression.     

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

Effect of metals on nucleoside hydroperoxide, a product of ionizing radiation in DNA

Ionizing radiation causes formation of thymine hydroperoxides in DNA. Their decomposition generates more stable products and active oxygen species which may oxidize other DNA bases. We have determined the effects of free and chelated metal ions on the degradation of 5-hydroperoxymethyl-2'-deoxyuridine (HPMdU). Two products were formed as analyzed by HPLC: 5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU). Sn(II) and Fe(II) caused instantaneous HPMdU degradation; Sn(II) generated only HMdU, whereas Fe(II) formed about equal amounts of both. Sn(IV) and Fe(III) were inactive. Cu(I), Cu(II), and Co(II) caused a time-dependent formation of both products, with FdU predominating. In the presence of Cu(I), Cu(II), and Fe(II), formate inhibited formation of HMdU but enhanced that of FdU. EDTA abolished Cu(I)-induced decomposition of HPMdU but only decreased that which was mediated by Cu(II). In contrast, EDTA enhanced the activity of Fe(III) with a time-dependent formation of FdU. EDTA and diethylenetriaminepentaacetic acid (DTPA) caused an instantaneous Fe(II)-mediated decomposition of HPMdU to FdU. Only desferal partially inhibited the activity of Fe(II), whereas the activities of Cu(I), Cu(II), and Fe(III) were blocked by desferal and DTPA. Possible mechanisms of HPMdU degradation by metal ions in the absence or presence of formate or chelators as well as formation of the .OH are discussed.     

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

The fluorescent intercalation complex of ethidium bromide with DNA was used as a probe to demonstrate damage in the base-pair region of DNA, due to the action of superoxide radicals. The O.2- radical itself, generated by gamma-radiolysis of oxygenated aqueous Na-formate solutions, is rather ineffective with respect to impairment of DNA. Copper(II) ions, known to interact with DNA by coordinate binding at purines, enhance the damaging effect of O.2-. Addition of H2O2 to the DNA/Cu(II) system gives rise to further enhancement, so that DNA impairment by O.2- becomes comparable to that initiated by .OH radicals. These results suggest that the modified, Cu(II)-catalysed, Haber-Weiss process transforms O.2- into .OH radicals directly at the target molecule, DNA-Cu2+ + O.2-----DNA-Cu+ + O2 DNA-Cu+ + H2O2----DNA...OH + Cu2+ + OH- in a "site-specific" mechanism as proposed for other systems (Samuni et al. 1981; Aronovitch et al. 1984). Slow DNA decomposition also occurs without gamma-irradiation by autocatalysis of DNA/Cu(II)/H2O2 systems. In this context we observed that Cu(II) in the DNA-Cu2+ complex (unlike free Cu2+) is capable of oxidizing Fe(II) to Fe(III), thus the redox potential of the Cu2+/Cu+ couple appears to be higher than that of the Fe3+/Fe2+ couple when the ions are complexed with DNA. Metal-catalysed DNA damage by O.2- also occurs with Fe(III), but not with Ag(I) or Cd(II) ions. It was also observed that Cu(II) ions (but neither Ag(I) nor Cd(II] efficiently quench the fluorescence of the intercalation complex of ethidium bromide with DNA.     

Manganese-mediated oxidative damage of cellular and isolated DNA by isoniazid and related hydrazines: non-Fenton-type hydroxyl radical formation.

The mechanism by which hydrazines induce damage to cellular and isolated DNA in the presence of metal ions has been investigated by pulsed-field gel electrophoresis (PFGE), DNA sequencing methods, and the ESR spin-trapping technique. For the detection of single-strand breaks by PFGE, an experimental procedure with alkali treatment has been designed. Isoniazid, hydrazine, and phenylhydrazine induced DNA single- and double-strand breaks in cells pretreated with Mn(II), whereas iproniazid did not. With isolated 32P-DNA, isoniazid produced DNA damage in the presence of Cu(II), Mn(II), or Mn(III). Iproniazid damage isolated DNA only in the presence of Cu(II). The Cu(II)-mediated DNA damage by isoniazid or iproniazid is due to active oxygen species other than hydroxyl free radical (.OH), presumably the Cu(I)-peroxide complex. Cleavage of isolated DNA by isoniazid plus Mn(II) occurred without marked site specificity. The DNA damage was inhibited by .OH scavengers and superoxide dismutase (SOD) but not by catalase, suggesting the involvement of .OH formed via O2- but not via H2O2. Consistently, in ESR experiments .OH formation was observed during Mn(II)-catalyzed autoxidation of isoniazid, and the .OH formation was inhibited by SOD, but not by catalase. Iproniazid plus Mn(II) produced no or little .OH. We propose a reaction mechanism for the .OH formation without a H2O2 intermediate during manganese-catalyzed autoxidation of hydrazine. The present and previous data raise the possibility that hydrazines plus Mn(II)-induced cellular DNA damage may occur, at least in part, through the non-Fenton-type reaction.     

Interactions between metal ions and carbohydrates: the coordination behavior of neutral erythritol to transition metal ions

The single crystals of coordinated complexes of neutral erythritol (C4H10O4) with various transition metal ions were synthesized and studied using FT-IR and single crystal X-ray diffraction analysis. Two CuCl2-erythritol complexes (denoted as CuE(I) and CuE(II)) were obtained. In CuE(I), Cu2+ coordinates with two chloride ions and four OH groups from two erythritol molecules. Two copper centers are linked by one erythritol molecule to form a zigzag chain. For CuE(II), each Cu2+ coordinates with two OH groups from an erythritol molecule and two chloride ions. The crystal of CuE(II) contains complexed and free erythritol, the dimers of [Cu2Cl4(C4H10O4)] further form a [Cu2Cl4(C4H10O4)]infinity chain via secondary Cu...Cl bonds, both the dimer unit of [Cu2Cl4.(C4H10O4)] and non-coordinated C4H10O4 unit exist side by side in the crystal. MnCl2-erythritol complex whose structure is similar to CuE(I) is also acquired. The OH groups of erythritol act as ligand to coordinate to metal ions on one hand, one the other hand, OH groups form hydrogen bonds network that link chain and layer together to build three-dimensional structures.     

The regeneration with ascorbate of Helix pomatia methaemocyanin mediated by hydrogen peroxide

The regeneration of the copper bands of H. pomatia haemocyanin proceeds much more slowly with an excess of ascorbate than with a slight excess of hydrogen peroxide. The regenerating agent with ascorbate is hydrogen perioxide, formed in its autoxidation at the air. This was concluded after regeneration experiments with ascorbate under strictly anaerobic conditions and at the air in the presence of catalase. The autoxidation of ascorbate was catalysed by Fe and Cu ions. In the presence of EDTA there is still metal catalysis, especially in slightly alkaline medium, due to the Fe(III)-EDTA complex. Addition of diethylenetriamine pentaacetate completely abolished the metal catalysis.     

Paradoxical role of lipid metabolism in heart function and dysfunction

The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single stranded M13 phage DNA. In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions. For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested [Fe(II), Co(II), Ni(II), Mn(II) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin were effective or less effecive than quercetin in causing DNA breakage. In the case of the quercetin-Cu(II) reaction, Cu(I) was shown to be essential intermediate by using the Cu(1)-sequestering reagent, bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions were reduced by one quercetin molecule; in contrast two ions were reduced per quercetin molecule in the DNA breakage reaction. Equally neocuproine inhibited the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case). The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation. From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.     

Biological activity of complexes derived from pyridine-2-carbaldehyde thiosemicarbazone. Structure of

Biological studies on [Fe(L)2](NO3).0.5H2O (1), [Fe(L)2][PF6] (2), [Co(L)2](NCS) (3), [Ni(HL)2]Cl2.3H2O (4) and Cu(L)(NO3) (5), where HL=C7H8N4S, pyridine-2-carbaldehyde thiosemicarbazone, have been carried out. The crystal structure of compound 3 has been solved. It consists of discrete monomeric cationic entities containing cobalt(III) ions in a distorted octahedral environment. The metal ion is bonded to one sulfur and two nitrogen atoms of each thiosemicarbazone molecule. The thiocyanate molecules act as counterions. The copper(II) and iron(III) complexes react with reduced glutathione and 2-mercaptoethanol. The reaction of compound 1 with the above thiols causes the reduction of the metal ion and bis(thiosemicarbazonato)iron(II) species are obtained. The redox activity, and in particular the reaction with cell thiols, seems to be related to the cytotoxicity of these complexes against Friend erithroleukemia cells and melanoma B16F10 cells.     

Preparation,characterization and crystal structures of manganese(II), iron(III) and copper(II) complexes of the bis[di-1,1-(2-pyridyl)ethyl]amine (BDPEA) ligand; evaluation of their DNA cleavage activities

The synthesis of a new tetrapyridyl ligand, bis[di-1,1-(2-pyridyl)ethyl]amine (BDPEA), is described. Complexation of this ligand with manganese(II), iron(III) or copper(II) chlorides afforded mononuclear complexes: Mn(BDPEA)Cl2 (1) [Fe (BDPEA)Cl2]Cl (2) and [Cu(BDPEA)Cl]Cl (3). In all cases, BDPEA is coordinated to the metal center by three pyridine nitrogen atoms and the secondary amine. The geometrical environments around the metals in Mn(BDPEA)Cl2 and [Fe(BDPEA)Cl2]Cl are best described as distorted octahedrals and in [Cu (BDPEA)Cl]Cl as a slightly distorted square pyramid. The DNA cleavage activities of manganese(II), iron (III) or copper(II) complexes of both BDPEA and another tetrapyridyl ligand, bis[di(2-pyridyl) methyl]amine (BDPMA), in the presence of an oxidant (H2O2) or a reducing agent (ascorbate) with air, are reported. The iron(III) complexes exhibited significantly enhanced efficiencies, compared to copper(II) complexes. [Fe(BDPEA)Cl2]Cl is found to be the most active DNA cleaver, in agreement with a better stability of BDPEA in oxidizing conditions.     

Metal ion-induced activation of molecular oxygen in pigmented polymers

Diamagnetic and paramagnetic metal ions enhanced the rate of production of hydrogen peroxide during autoxidation of melanin pigments, as measured using an oxidase electrode. However, redox-active metal ions, such as Fe3+ and Cu2+, caused a marked decrease in H2O2 production. Evidence for redox-active metal ion-dependent formation of hydroxyl radicals during autoxidation of melanin pigments has been obtained using the electron spin resonance-spin trapping method. Evidence for direct reduction of Fe3+ by melanin polymers also has been obtained using optical spectroscopy. Mechanisms of molecular activation of oxygen induced by metal ions on melanin polymers are discussed.     

DNA damage and 2'-deoxyguanosine oxidation induced by S(IV) autoxidation catalyzed by copper(II) tetraglycine complexes: synergistic effect of a second metal ion

S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).     

Spectroscopic analysis of the unfolding of transition metal-ion complexes of human lactoferrin and transferrin.

1. Human lactoferrin and transferrin are capable of binding several transition metal ions [Fe(III), Cu(II), Mn(III), Co(III)] into specific binding sites in the presence of bicarbonate. 2. Increased conformational stability and increased resistance to protein unfolding is observed for these metal-ion complexes compared to the apoprotein form of these proteins. 3. Mn(III)-lactoferrin and transferrin complexes exhibit steeper denaturation transitions than the Co(III) complexes of these proteins suggesting greater cooperativity in the unfolding process. 4. The incorporation of Fe(III) into the specific metal binding sites offers the greatest resistance to thermal unfolding when compared to the other transition metal ions studied. 5. Non-coincidence of unfolding transitions is observed, with fluorescence transition midpoints being lower than those determined by absorbance measurements. 6. Fully denatured proteins in the presence of urea and alkyl ureas exhibit fluorescence wavelength maxima at 355-356 nm indicative of tryptophan exposure upon protein unfolding.