In vitro interaction between homocysteine and copper ions: Potential redox implications

Homocysteine (Hcys) has been implicated in various oxidative stress-related disorders. The presence of a thiol on its structure allows Hcys to exert a double-edge redox action. Depending on whether Cu2+ ions occur concomitantly, Hcys can either promote or prevent free radical generation and its consequences. We have addressed in vitro the interaction between Hcys and Cu2+ ions, in terms of the consequences that such interaction may have on the free radical scavenging properties of Hcys and on the redox state and redox activity of the metal. To this end, we investigated the free radical-scavenging, O2(*-)-generating, and ascorbate-oxidizing properties of the interacting species by assessing the bleaching of ABTS*+ radicals, the reduction of O2(*-)-dependent cytochrome c, and the copper-dependent oxidation of ascorbate, respectively. In addition, electron paramagnetic resonance and Cu(I)-bathocuproine formation were applied to assess the formation of paramagnetic complexes and the metal redox state. Upon a brief incubation, the Hcys/Cu2+ interaction led to a decrease in the free radical-scavenging properties of Hcys, and to a comparable loss of the thiol density. Both effects were partial and were not modified by increasing the incubation time, despite the presence of Cu2+ excess. Depending on the molar Hcys:Cu2+ ratio, the interaction resulted in the formation of mixtures that appear to contain time-stable and ascorbate-reducible Cu(II) complexes (for ratios up to 2:1), and ascorbate- and oxygen-redox-inactive Cu(I) complexes (for ratios up to 4:1). Increasing the interaction ratio beyond 4:1 was associated with the sudden appearance of an O2(*-)-generating activity. The data indicate that depending on the molar ratio of interaction, Hcys and Cu2+ react to form copper complexes that can promote either antioxidant or pro-oxidant actions. We speculate that the redox activity arising from a large molar Hcys excess may partially underlie the association between hyper-homocysteinemia and a greater risk of developing oxidative-related cardiovascular diseases.     

Anti-oxidant and pro-oxidant behaviour of bucillamine.

Bucillamine (BUC) is used clinically for the treatment of rheumatoid arthritis. Some of the pharmacological action of BUC has been reported as being dependent on the production of reactive oxygen species (ROS). In this paper the reactivity of BUC with superoxide anion radical (O(2) (*-)) generated from potassium superoxide/18-crown-6 ether dissolved in DMSO, hydroxyl radical (HO(*)) produced in the Cu(2+)-H(2)O(2) reaction, peroxyl radical (ROO(*)) from 2,2'-azobis (2-amidino-propane) dichloride decomposition, and singlet oxygen ((1)O(2)) from a mixture of alkaline aqueous H(2)O(2) and acetonitrile, have been investigated. Chemiluminescence, fluorescence, electron paramagnetic resonance (EPR) spin-trapping techniques and the deoxyribose and oxygen radical absorbance capacity towards ROO(*) (ORAC(ROO)) assays were used to elucidate the anti- and pro-oxidative behaviours of BUC towards ROS. The results indicated that BUC efficiently inhibited chemiluminescence from the O(2) (*-)-generating system at relatively high concentrations (0.5-2 mmol/L); however, at lower concentrations (<0.5 mmol/L) the drug enhanced light emission. The behaviour of BUC was correlated with a capacity to decrease the chemiluminescence signal from the Cu(2+)-H(2)O(2) system; scavenging HO(*) was effective only at high concentrations (1-2 mmol/L) of the drug. Bucillamine also prevented deoxyribose degradation induced by HO(*) in a dose-dependent manner, reaching maximal inhibition (24.5%) at a relative high concentration (1.54 mmol/L). Moreover, BUC reacts with ROO(*); the relative ORAC(ROO) was found to be 0.34 micromol/L Trolox equivalents/micromol sample. The drug showed quenching of (1)O(2)-dependent 2,2,6,6-tetramethylpiperidine-N-oxide radical formation from 2,2,6,6-tetramethyl-piperidine (e.g. 90% inhibition was found at 1 mmol/L concentration). The results showed that BUC may directly scavenge ROS or inhibit reactions generating them. However, the drug may have pro-oxidant activity under some reaction conditions.     

A copper chelate of thiosemicarbazone NSC 689534 induces oxidative/ER stress and inhibits tumor growth in vitro and in vivo

In this study, a Cu(2+) chelate of the novel thiosemicarbazone NSC 689534 was evaluated for in vitro and in vivo anti-cancer activity. Results demonstrated that NSC 689534 activity (low micromolar range) was enhanced four- to fivefold by copper chelation and completely attenuated by iron. Importantly, once formed, the NSC 689534/Cu(2+) complex retained activity in the presence of additional iron or iron-containing biomolecules. NSC 689534/Cu(2+) mediated its effects primarily through the induction of ROS, with depletion of cellular glutathione and protein thiols. Pretreatment of cells with the antioxidant N-acetyl-l-cysteine impaired activity, whereas NSC 689534/Cu(2+) effectively synergized with the glutathione biosynthesis inhibitor buthionine sulfoximine. Microarray analysis of NSC 689534/Cu(2+)-treated cells highlighted activation of pathways involved in oxidative and ER stress/UPR, autophagy, and metal metabolism. Further scrutiny of the role of ER stress and autophagy indicated that NSC 689534/Cu(2+)-induced cell death was ER-stress dependent and autophagy independent. Last, NSC 689534/Cu(2+) was shown to have activity in an HL60 xenograft model. These data suggest that NSC 689534/Cu(2+) is a potent oxidative stress inducer worthy of further preclinical investigation.     

Direct observation of trapping and release of nitric oxide by glutathione and cysteine with electron paramagnetic resonance spectroscopy

While the biosynthesis of nitric oxide (NO) is well established, one of the key issues that remains to be solved is whether NO participates in the biological responses right after generation through biosynthesis or there is a "secret passage" via which NO itself is trapped, transported, and released to exert its functions. It has been shown that NO reacts with thiol-containing biomolecules (RSH), like cysteine (Cys), glutathione (GSH), etc., to form S-nitrosothiols (RSNOs), which then release nitrogen compounds, including NO. The direct observation of trapping of NO and its release by RSNO has not been well documented, as most of the detection techniques measure the content of NO as well as nitrite and nitrate. Here we use spin-trapping electron paramagnetic resonance (EPR) technique to measure NO content directly in the reaction time course of samples of GSH and Cys ( approximately mM) mixed with NO ( approximately microM) in the presence of metal ion chelator, which pertains to physiological conditions. We demonstrate that NO is readily trapped by these thiols in less than 10 min and approximately 70-90% is released afterward. These data imply that approximately 10-30% of the reaction product of NO does not exist in the free radical form. The NO release versus time curves are slightly pH dependent in the presence of metal ion chelator. Because GSH and Cys exist in high molar concentrations in blood and in mammalian cells, the trapping and release passage of NO by these thiols may provide a mechanism for temporal and spatial sequestration of NO to overcome its concentration gradient-dependent diffusion, so as to exert its multiple biological effects by reacting with various targets through regeneration.     

Inhibition by thiols of copper(II)-induced oxidation of oxyhemoglobin.

The ability of thiols, 2-imidazolethiones and uric acid to protect bovine oxyhemoglobin from copper(II)-induced oxidation to methemoglobin was investigated. The oxidation of oxyhemoglobin by Cu(II) proceeded in two phases: (1) an initial rapid reaction (less than 30 s) followed by (2) a slower reaction that carried it to completion. Thiols, including N-acetyl-L-cysteine, DL-dithiothreitol, reduced glutathione, DL-homocysteine, 2-mercaptoethanol and 2- and 3-mercaptopropionic acid, whose sulfhydryl groups were slowly oxidized by Cu(II) (with the exception of 2-mercaptopropionic acid), protected oxyhemoglobin in both phases of the reaction. Other thiols, including L-cysteine, cysteamine, and D-penicillamine, whose sulfhydryl groups were readily oxidized by Cu(II), protected hemoglobin initially, but within 2-4 min, the rate of methemoglobin formation was the same as Cu(II)-treated oxyhemoglobin. 2-Mercaptoimidazole and 1-methyl-2-mercaptoimidazole, which complex Cu(II) and inhibit Cu(II)-catalyzed oxidation of ascorbic acid, also protected hemoglobin in the initial phase, but not in the second phase. Uric acid, L-ergothioneine, and thiourea did not protect oxyhemoglobin in either the fast or slow phase. Cu(II) may have a coordination site involved in the oxidation of hemoglobin that is not blocked by the 2-imidazolethiones, uric acid, or the oxidized thiols. It is concluded that certain thiols that complex Cu(II) and are not rapidly oxidized will protect oxyhemoglobin from Cu(II)-induced oxidation, but the thiols are no longer effective once they are oxidized.     

Electron paramagnetic resonance study of the active site of copper-substituted human glyoxalase I

Zn2+ in native glyoxalase I from human erythrocytes can be replaced by Cu2+, giving an inactive enzyme. Cu2+ was demonstrated to compete with the activating metals Zn2+ and Mn2+, indicating a common binding site on the enzyme for these metal ions. The electron paramagnetic resonance (EPR) spectra of 63Cu(II) glyoxalase I at 77 K and of its complexes with glutathione and some glutathione derivatives are characteristic of Cu2+ in an elongated octahedral coordination (g parallel = 2.34, g perpendicular = 2.09, and A parallel = 14.2 mT). The low-field bands of the free enzyme are asymmetric and become symmetrical upon addition of glutathione or S-(p-bromobenzyl)glutathione but not S-(D-lactoyl)glutathione. The results indicate the existence of two conformations of Cu(II) glyoxalase I, in agreement with the effects caused by these compounds on the protein fluorescence. The copper hyperfine line at low field in the EPR spectrum of the S-(p-bromobenzyl)glutathione complex of 63Cu(II) glyoxalase I shows a triplet structure, indicative of coupling to one nitrogen ligand in the equatorial plane. Similar results were obtained with the glutathione complex. By addition of the spectrum of the S-(p-bromobenzyl)glutathione complex and a spectrum corresponding to two nitrogen ligands with two different coupling constants, a good fit was obtained for the low-field region of the asymmetric spectrum of free 63Cu(II) glyoxalase I. The first two spectra are assumed to correspond to two separate conformational states of the enzyme. The results demonstrate that at least one nitrogen ligand is involved in the binding of Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Classes of thiols that influence the activity of the skeletal muscle calcium release channel

The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) is a prototypic redox-responsive ion channel. Nearly half of the 101 cysteines per RyR1 subunit are kept in a reduced (free thiol) state under conditions comparable with resting muscle. Here we assessed the effects of physiological determinants of cellular redox state (oxygen tension, reduced (GSH) or oxidized (GSSG) glutathione, and NO/O(2) (released by 3-morpholinosydnonimine)) on RyR1 redox state and activity. Oxidation of approximately 10 RyR1 thiols (from approximately 48 to approximately 38 thiols/RyR1 subunit) had little effect on channel activity. Channel activity increased reversibly as the number of thiols was further reduced to approximately 23/subunit, whereas more extensive oxidation (to approximately 13 thiols/subunit) inactivated the channel irreversibly. Neither S-nitrosylation nor tyrosine nitration contributed to these effects. The results identify at least three functional classes of RyR1 thiols and suggest that 1) the channel may be protected from oxidation by a large reservoir of functionally inert thiols, 2) the channel may be designed to respond to moderate oxidative stress by a change in activation setpoint, and 3) the channel is susceptible to oxidative injury under more extensive conditions.     

Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.     

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.     

Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer's disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1-28): insights from a range of complementary spectroscopic techniques

There is now direct evidence that copper is bound to amyloid-beta peptide (Abeta) in senile plaque of Alzheimer's disease. Copper is also linked with the neurotoxicity of Abeta and free radical damage, and Cu(2+) chelators represent a possible therapy for Alzheimer's disease. We have therefore used a range of complementary spectroscopies to characterize the coordination of Cu(2+) to Abeta in solution. The mode of copper binding is highly pH-dependent. EPR spectroscopy indicates that both coppers have axial, Type II coordination geometry, square-planar or square-pyramidal, with nitrogen and oxygen ligands. Circular dichroism studies indicate that copper chelation causes a structural transition of Abeta. Competition studies with glycine and l-histidine indicate that copper binds to Abeta-(1-28) at pH 7.4 with an affinity of K(a) approximately 10(7) m(-1). (1)H NMR indicates that histidine residues are involved in Cu(2+) coordination but that Tyr(10) is not. Studies using analogues of Abeta-(1-28) in which each of the histidine residues have been replaced by alanine or in which the N terminus is acetylated suggest that the N terminus and His(13) are crucial for Cu(2+) binding and that His(6) and His(14) are also implicated. Evidence for the link between Alzheimer's disease and Cu(2+) is growing, and our studies have made a significant contribution to understanding the mode of Cu(2+) binding to Abeta in solution.     

N-Terminal deletions modify the Cu2+ binding site in amyloid-beta

Copper is implicated in the in vitro formation and toxicity of Alzheimer's disease amyloid plaques containing the beta-amyloid (Abeta) peptide (Bush, A. I., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 11934). By low temperature electron paramagnetic resonance (EPR) spectroscopy, the importance of the N-terminus in creating the Cu(2+) binding site in native Abeta has been examined. Peptides that contain the proposed binding site for Cu(2+)-three histidines (H6, H13, and H14) and a tyrosine (Y10)-but lack one to three N-terminal amino acids, do not bind Cu(2+) in the same coordination environment as the native peptide. EPR spectra of soluble Abeta with stoichiometric amounts of Cu(2+) show type 2 Cu(2+) EPR spectra for all peptides. The ligand donor atoms to Cu(2+) are 3N1O when Cu(2+) is bound to any of the Abetapeptides (Abeta16, Abeta28, Abeta40, and Abeta42) that contain the first 16 amino acids of full-length Abeta. When a Y10F mutant of Abeta is used, the coordination environment for Cu(2+) remains 3N1O and Cu(2+) EPR spectra of this mutant are identical to the wild-type spectra. Isotopic labeling experiments show that water is not the O-atom donor to Cu(2+) in Abeta fibrils or in the Y10F mutant. Further, we find that Cu(2+) cannot be removed from Cu(2+)-containing fibrils by washing with buffer, but that Cu(2+) binds to fibrils initially assembled without Cu(2+) in the same coordination environment as in fibrils assembled with Cu(2+). Together, these results indicate (1) that the O-atom donor ligand to Cu(2+) in Abeta is not tyrosine, (2) that the native Cu(2+) binding site in Abeta is sensitive to small changes at the N-terminus, and (3) that Cu(2+) binds to Abetafibrils in a manner that permits exchange of Cu(2+) into and out of the fibrillar architecture.     

Role of Amino Thiols in Luminol Chemiluminescence Coupled with Copper(II)-catalysed Oxidation of Cysteine and Glutathione

The Cu(II)-catalysed oxidation of the amino thiols cysteine and glutathione with oxygen was examined in the presence of luminol. Light emission was markedly suppressed when the concentration of amino thiols was greater than that of Cu(II). Cysteine caused a delay of chemiluminescence (CL) and no CL emission was observed at high concentrations of GSH. The suppression in CL could be explained in terms of the complexation of the Cu(II) catalyst by formation of a Cu(II)–dicysteine complex in case of cysteine, and of a Cu(II)–GSSG complex in case of GSH.     

Effects of copper and zinc ions on photosystem II studied by EPR spectroscopy.

The effect of Zn(2+) or Cu(2+) ions on Mn-depleted photosystem II (PS II) has been investigated using EPR spectroscopy. In Zn(2+)-treated and Cu(2+)-treated PS II, chemical reduction with sodium dithionite gives rise to a signal attributed to the plastosemiquinone, Q(A)(*)(-), the usual interaction with the non-heme iron being lost. The signal was identified by Q-band EPR spectroscopy which partially resolves the typical g-anisotropy of the semiquinone anion radical. Illumination at 200 K of the unreduced samples gives rise to a single organic free radical in Cu(2+)-treated PS II, and this is assigned to a monomeric chlorophyll cation radical, Chl a(*)(+), based on its (1)H-ENDOR spectrum. The Zn(2+)-treated PS II under the same conditions gives rise to two radical signals present in equal amounts and attributed to the Chl a(*)(+) and the Q(A)(*)(-) formed by light-induced charge separation. When the Cu(2+)-treated PS II is reduced by sodium ascorbate, at >/=77 K electron donation eliminates the donor-side radical leaving the Q(A)(*)(-) EPR signal. The data are explained as follows: (1) Cu(2+) and Zn(2+) have similar effects on PS II (although higher concentrations of Zn(2+) are required) causing the displacement of the non-heme Fe(2+). (2) In both cases chlorophyll is the electron donor at 200 K. It is proposed that the lack of a light-induced Q(A)(*)(-) signal in the unreduced Cu(2+)-treated sample is due to Cu(2+) acting as an electron acceptor from Q(A)(*)(-) at low temperature, forming the Cu(+) state and leaving the electron donor radical Chl a(*)(+) detectable by EPR. (3) The Cu(2+) in PS II is chemically reducible by ascorbate prior to illumination, and the metal can therefore no longer act as an electron acceptor; thus Q(A)(*)(-) is generated by illumination in such samples. (4) With dithionite, both the Cu(2+) and the quinone are reduced resulting in the presence of Q(A)(*)(-) in the dark. The suggested high redox potential of Cu(2+) when in the Fe(2+) site in PS II is in contrast to the situation in the bacterial reaction center where it has been shown in earlier work that the Cu(2+) is unreduced by dithionite. It cannot be ruled out however that Q(A)-Cu(2+) is formed and a magnetic interaction is responsible for the lack of the Q(A)(-) signal when no exogenous reductant is present. With this alternative possibility, the effects of reductants would be explained as the loss of Cu(2+) (due to formation of Cu(+)) leading to loss of the Cu(2+) from the Fe(2+) site due to the binding equilibrium. The quite different binding and redox behavior of the metal in the iron site in PS II compared to that of the bacterial reaction center is presumably a further reflection of the differences in the coordination of the iron in the two systems.     

Cu(III)-Polypeptide complexes exhibiting SOD-like activity

Summary The SOD-like activity of Cu(III) -complexes with polypeptides poly-L-lysine and poly-L-glutamic acid respectively was investigated. The Cu(II)-polypeptide complexes were first oxidized by K₂IrCl₆ to give the corresponding Cu(III) -compounds.The oxidation of Cu(II) and the corresponding Cu(II)/Cu(III) potential was evaluated by cyclic voltammetry (c.v.), UV-Vis and EPR spectroscopic (r.t.) experiments. Spin trapping EPR spectra were also conducted to confirm the formation of the superoxide radical. The SOD-like activity of each Cu(III)-complex was proved using the nitro blue tetrazolium (NBT) method slightly modified.     

The reactivity of thiols and disulfides with different redox states of myoglobin. Redox and addition reactions and formation of thiyl radical intermediates.

The reactivity of several thiols, including glutathione, dihydrolipoic acid, cysteine, N-acetyl cysteine, and ergothioneine, as well as several disulfides, toward different redox states of myoglobin, mainly met-myoglobin (HX-FeIII) and ferrylmyoglobin (HX-FeIV=O), was evaluated by optical spectral analysis, product formation, and thiyl free radical generation. Only dihydrolipoic acid reduced met-myoglobin to oxy-myoglobin, whereas all the other thiols tested did not interact with met-myoglobin. Although the redox transitions involved in the former reduction were expected to yield the dihydrolipoate thiyl radical, the reaction was EPR silent. Conversely, all thiols interacted to different extent with the high oxidation state of myoglobin, i.e. ferrylmyoglobin, via two processes. First, direct electron transfer to heme iron in ferrylmyoglobin (HX-FeIV=O) with formation of met-myoglobin (HX-FeIII) or oxymyoglobin (HX-FeIIO2); the former transition was effected by all thiols except dihydrolipoate, which facilitated the latter, i.e. the formation of the two-electron reduction product of ferrylmyoglobin. Second, nucleophilic addition onto a pyrrole in ferrylmyoglobin with subsequent formation of sulfmyoglobin. The contribution of either direct electron transfer to the heme iron or nucleophilic addition depended on the physicochemical properties of the thiol involved and on the availability of H2O2 to reoxidize met-myoglobin to ferrylmyoglobin. The thiyl radicals of glutathione, cysteine, and N-acetylcysteine were formed during the interaction of the corresponding thiols with ferrylmyoglobin and detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyroline-N-oxide. The intensity of the EPR signal was insensitive to superoxide dismutase and it was decreased, but not suppressed, by catalase. The disulfides of glutathione and cysteine did not react with ferrylmyoglobin, but the disulfide bridge in lipoic acid interacted efficiently with the ferryl species by either reducing directly the heme iron to form met-myoglobin or adding onto a pyrrole ring to form sulfmyoglobin; either process depended on the presence or absence of catalase (to eliminate the excess of H2O2) in the reaction mixture, respectively. The biological significance of the above results is discussed in terms of the occurrence and distribution of high oxidation states of myoglobin, its specific participation in cellular injury, and its potential interaction with biologically important thiols leading to either recovery of myoglobin or generation of nonfunctional forms of the hemoprotein as sulfmyoglobin.     

Radioprotection of DNA in isolated nuclei by naturally occurring thiols at intermediate oxygen tension.

Incubation of isolated Chinese hamster ovary cell nuclei, equilibrated in an atmosphere containing 2% O2, with glutathione, cysteine, or cysteamine resulted in a decrease in the number of X-ray-induced DNA double-strand breaks (DSBs), determined by pH 9.0 filter elution. In the absence of exogenous thiol, no sensitization was observed with the addition of N-ethylmaleimide, indicating that endogenous thiols were not present at significant levels. Protection by 0.3 mM glutathione was not enhanced by the addition of exogenous glutathione S-transferases or by glutathione peroxidase. The data were analyzed according to a simple competition model with various hypotheses. Cysteamine was more than an order of magnitude more effective than the other thiols tested, on a molar basis, in preventing DSB formation. Depending on the hypothesis used to evaluate the data, glutathione was either much less effective, on a molar basis, in preventing the bulk of the DSBs or was capable of preventing only approximately 55% of the damage, regardless of concentration. These data suggest that natural thiols other than glutathione may contribute to cellular radioprotection even if their concentration is much lower than that of glutathione. The data also suggest that despite the relative inefficiency of glutathione as a radioprotector, some areas of oxygenated tissues--where the oxygen tension falls below 2%--may be protected by glutathione concentrations in the physiological range of 3-20 mM.     

Effects of redox buffer properties on the folding of a disulfide-containing protein: dependence upon pH, thiol pKa, and thiol concentration

Aliphatic thiols are effective as redox buffers for folding non-native disulfide-containing proteins into their native state at high pH values (8.0-8.5) but not at neutral pH values (6-7.5). In developing more efficient and flexible redox buffers, a series of aromatic thiols was analyzed for its ability to fold scrambled ribonuclease A (sRNase A). At equivalent pH values, the aromatic thiols folded sRNase A 10-23 times faster at pH 6.0, 7-12 times faster at pH 7.0, and 5-8 times faster at pH 7.7 than the standard aliphatic thiol glutathione. Similar correlations between thiol pK(a) values and folding rates at each pH value suggest that the apparent folding rate constants (k(app)) are a function of the redox buffer properties (pH, thiol pK(a) and [RSH]). Fitting the observed data to a three-variable model (logk(app)=-4.216(+/-0.030)+0.5816(+/-0.0036)pH-0.233(+/-0.004)pK(a)+log(1-e(-0.98(+/-0.02)[RSH]))) gave good statistics: r2=0.915, s=0.10.     

Tocopherylquinone and tocopherylhydroquinone

Tocopherylquinone (TQ) is formed in the antioxidant action of tocopherol (T). TQ was found in human subjects and it was observed that the ratio alphaTQ/ alphaT increased in general with increasing oxidative stress. TQ is reduced to tocopheryl hydroquinone (TQH2) but the ratio TQH2/TQ in vivo has not been reported. TQH2 acts as a potent radical-scavenging antioxidant. alphaTQH2 is more reactive toward radicals than ubiquinol, a reduced form of coenzyme Q, and alphaT. The overall efficacy of TQH2 as an antioxidant is determined by the fate of semiquinone radical formed from TQH2 as well as the reactivity toward oxygen radicals. Partly substituted gammaTQ, but not alphaTQ, exerts cytotoxicity by both redox cycling and reaction with protein thiols and glutathione.     

An ESR investigation of the reactions of glutathione, cysteine and penicillamine thiyl radicals: competitive formation of RSO., R., RSSR-., and RSS(.)

The reactions of the cysteine, glutathione and penicillamine thiyl radicals with oxygen and their parent thiols in frozen aqueous solutions have been elucidated through electron spin resonance spectroscopy. The major sulfur radicals observed are: (1) thiyl radicals, RS.; (2) disulfide radical anions. RSSR-.; (3) perthiyl radicals, RSS. and upon introduction of oxygen; (4) sulfinyl radicals, RSO., where R represents the remainder of the cysteine, glutathione or penicillamine moiety. The radical product observed depends on the pH, concentration of thiol, and presence or absence of molecular oxygen. We find that the sulfinyl radical is a ubiquitous intermediate in the free radical chemistry of these important biological compounds, and also show that peroxyl radical attack on thiols may lead to sulfinyl radicals. We elaborate the observed reaction sequences that lead to sulfinyl radicals, and, using 17O isotopic substitution studies, demonstrate that the oxygen atom in sulfinyl radicals originates from dissolved molecular oxygen. In addition, the glutathione thiyl radical is found to abstract hydrogen from the alpha-carbon position on the cysteine residue of glutathione to form a carbon-centered radical.     

Copper-specific damage in human erythrocytes exposed to oxidative stress

Ascorbate and complexes of Cu(II) and Fe(III) are capable of generating significant levels of oxygen free radicals. Exposure of erythrocytes to such oxidative stress leads to increased levels of methemoglobin and extensive changes in cell morphology. Cu(II) per mole is much more effective than Fe(III). However, isolated hemoglobin is oxidized more rapidly and completely by Fe(III)- than by Cu(II)-complexes. Both Fe(III) and Cu(II) are capable of inhibiting a number of the key enzymes of erythrocyte metabolism. The mechanism for the enhanced activity of Cu(II) has not been previously established. Using intact erythrocytes and hemolysates we demonstrate that Cu(II)-, but not Fe(III)-complexes in the presence of ascorbate block NADH-methemoglobin reductase. Complexes of Cu(II) alone are not inhibitory. The relative inability of Fe(III)-complexes and ascorbate to cause methemoglobin accumulation is not owing to Fe(III) association with the membrane, or its failure to enter the erythrocytes. The toxicity of Cu(II) and ascorbate appears to be a result of site-specific oxidative damage of erythrocyte NADH-methemoglobin reductase and the enzyme's subsequent inability to reduce the oxidized hemoglobin.