DNA strand scission by hemin-thiolate complexes as models of cytochrome P-450

Hemin-thiolate complexes, as chemical models for cytochrome P-450 monooxygenases, have been shown to cause strand scission of DNA. Circular super-coiled DNA was degraded to open-circular and linear forms by these complexes in 30 min at pH 7.8 under aerobic conditions, the degradation depending on the structure of the thiol ligand and the ratio of thiol ligand to hemin concentration. The relationship between the structure of the thiol ligand and DNA strand cleaving activity was examined. Complete cleavage of DNA was observed by complexes containing TGE and ME at 400-600 moles excess of thiol ligand to hemin, those containing Cys, CysMe, and CysEt at 50-200 moles excess, and those containing MPG, GSH, penta- and nona-peptides at 5-20 moles excess. Complexes containing NACys and MEA caused no cleavage of DNA. Inhibition experiments suggested the involvement of active oxygen species in the cleavage.     

Dietary isothiocyanate-induced apoptosis via thiol modification of DNA topoisomerase IIα

Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.     

Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands

The complexation of glutathione and related ligands by the nitrilotriacetic acid complex of Cd2+ (Cd(NTA)-) has been investigated by 1H NMR as a model for the coordination chemistry of Cd2+ and GSH in biological systems. Related ligands included glycine, glutamic acid, cysteine, N-acetylcysteine, penicillamine, N-acetylpenicillamine, mercaptosuccinic acid, and the S-methyl derivative of glutathione. The nature of the complexes formed was deduced from 1H NMR spectra of Cd(NTA)- and the ligands. Mixed ligand complexes (Cd(NTA)L) and single ligand complexes (CdLx) are formed with the thiol ligands, whereas only mixed ligand complexes form with glycine, glutamic acid and S-methylglutathione. Formation constants of the mixed and the single ligand complexes were determined from NMR data. The results indicate that formation constants for binding of a thiolate donor group by Cd2+, either as the free ion or in a coordinately unsaturated complex, are in the range 10(5)-10(6).     

Mechanism of Copper-Catalyzed Oxidation of Glutathione

The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal *OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and *OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.     

Properties and utility of the peculiar mixed disulfide in the bacterial glutathione transferase B1-1

Bacterial glutathione transferases appear to represent an evolutionary link between the thiol:disulfide oxidoreductase and glutathione transferase superfamilies. In particular, the observation of a mixed disulfide in the active site of Proteus mirabilis glutathione transferase B1-1 is a feature that links the two families. This peculiar mixed disulfide between Cys10 and one GSH molecule has been studied by means of ESR spectroscopy, stopped-flow kinetic analysis, radiochemistry, and site-directed mutagenesis. This disulfide can be reduced by dithiothreitol but even a thousand molar excess of GSH is poorly effective due to an unfavorable equilibrium constant of the redox reaction (K(eq) = 2 x 10(-4)). Although Cys10 is partially buried in the crystal structure, in solution it reacts with several thiol reagents at a higher or comparable rate than that shown by the free cysteine. Kinetics of the reaction of Cys10 with 4,4'-dithiodipyridine at variable pH values is consistent with a pK(a) of 8.0 +/- 0.1 for this residue, a value about 1 unit lower than that of the free cysteine. The 4,4'-dithiodipyridine-modified enzyme reacts with GSH in a two-step mechanism involving a fast precomplex formation, followed by a slower chemical step. The natural Cys10-GSH mixed disulfide exchanges rapidly with free [3H]GSH in a futile redox cycle in which the bound GSH is continuously replaced by the external GSH. Our data suggest that the active site of the bacterial enzyme has intermediate properties between those of the recently evolved glutathione transferases and those of the thiol:disulfide oxidoreductase superfamily.     

Submicrodeterminations of thiols, disulphides and thiol esters in serum by using o-hydroxymercuribenzoic acid and dithiofluorescein

1. Methods are described for selective estimation of thiols, disulphides and thiol esters in standard solutions and in serum. The methods are based on the reaction with the excess of o-hydroxymercuribenzoic acid (HMB) in alkaline solution with subsequent addition of dithiofluorescein in excess and determination of the extinction at 588mmu. The sensitivity of the methods amounts to 1.5x10(-9)g.equiv. in 5ml. of final solution. Of results obtained on standard solutions 80% have the errors within the range +/-4%. 2. It has been found that serum contains an unidentified substance (substance X) producing green complexes with dithiofluorescein which undergo decomposition on addition of formaldehyde. The correction for substance X must be estimated in a separate sample and taken into account. The concentration of substance X can be calculated from extinctions measured at 588mmu and 635mmu in the presence of dithiofluorescein in excess. 3. The selective determination of thiols and disulphides is based on different reaction rates with formaldehyde. The complexes between HMB and cysteine can be selectively decomposed by formaldehyde, and free glutathione can be selectively removed by formaldehyde in the presence of protein thiols. 4. Thiols are determined in the presence of triethylamine, thiols plus disulphides in the presence of triethylamine and sulphite, and thiols plus thiol esters in the presence of dimethylamine, with subsequent addition of ammonium sulphate.     

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma.

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 microM dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (less than 2 pmol) to detect the various forms of the four thiol compounds in human plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.     

Homocysteine effects on cellular glutathione peroxidase (GPx-1) activity under in vitro conditions

Hyperhomocysteinemia is associated with a lot of diseases including cardiovascular diseases and neural tube defect, but it has not been clarified exactly which mechanism is responsible for occurence disease. Here, homocysteine (Hcy) and cysteine (Cys), which are thiol containing amino acids, were examined for their effect on glutathione peroxidase (GPx) activity. It was observed that the GPx-1 activity was inhibited under severe hyperhomocysteinemia (50-500 microM Hcy) conditions, especially at low glutathione concentrations but that cysteine increased GPx-1 activity at low glutathione concentrations and inhibition clearly appeared at 500 microM Cys concentration.     

Interaction of hemin with placental glutathione transferase

To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.     

Macrophages regulate intracellular glutathione levels of lymphocytes. Evidence for an immunoregulatory role of cysteine

Macrophages consume cystine and generate approximately equivalent amounts of acid-soluble thiol. Stimulation of macrophages with bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF) strongly augments the amount of thiol released into the culture supernatant. Cysteine constitutes most of the acid-soluble thiol. The intracellular glutathione level and the DNA synthesis activity in mitogenically stimulated lymphocytes are strongly increased by either exogenously added cysteine, or (syngeneic) macrophages. This cysteine dependency is observed even in the presence of relatively high extracellular cystine concentration as they occur in the blood plasma. The extracellular cysteine concentration also has a strong influence on the intracellular glutathione concentration, viability, and DNA synthesis of cycling T cell clones. Moreover, the cysteine concentration in the culture medium on Day 3 and Day 4 of a 5-day allogeneic mixed lymphocyte culture (i.e., in the late phase of incubation) has a strong influence on the generation of cytotoxic T cell activity, indicating that regulatory effects of cysteine are not restricted to the early phase of the blastogenic response. The inhibitory effect of cysteine starvation on the DNA synthesis of the T cell clones and on the activation of cytotoxic T lymphocytes can be explained essentially by the depletion of intracellular glutathione, since similar effects are observed after treatment with buthionine sulfoximine (BSO), a specific inhibitor of the glutathione biosynthesis. BSO has practically no influence, however, on the N alpha-benzyloxycarbonyl Ne-t-butyloxycarbonyl-L-lysine-thiobenzyl-ester (BLT)-esterase activity and hemolytic activity of the cell lysates from cytotoxic T cells against sheep red blood cells (perforin activity). Taken together, our experiments indicate that cysteine has a regulatory role in the immune system analogous to the hormone-like lymphokines and cytokines. It is released by macrophages at a variable and regulated rate and regulates immunologically relevant functions of lymphocytes in the vicinity.     

Saccharomyces cerevisiae cells have three Omega class glutathione S-transferases acting as 1-Cys thiol transferases

The Saccharomyces cerevisiae genome encodes three proteins that display similarities with human GSTOs (Omega class glutathione S-transferases) hGSTO1-1 and hGSTO2-2. The three yeast proteins have been named Gto1, Gto2 and Gto3, and their purified recombinant forms are active as thiol transferases (glutaredoxins) against HED (beta-hydroxyethyl disulphide), as dehydroascorbate reductases and as dimethylarsinic acid reductases, while they are not active against the standard GST substrate CDNB (1-chloro-2,4-dinitrobenzene). Their glutaredoxin activity is also detectable in yeast cell extracts. The enzyme activity characteristics of the Gto proteins contrast with those of another yeast GST, Gtt1. The latter is active against CDNB and also displays glutathione peroxidase activity against organic hydroperoxides such as cumene hydroperoxide, but is not active as a thiol transferase. Analysis of point mutants derived from wild-type Gto2 indicates that, among the three cysteine residues of the molecule, only the residue at position 46 is required for the glutaredoxin activity. This indicates that the thiol transferase acts through a monothiol mechanism. Replacing the active site of the yeast monothiol glutaredoxin Grx5 with the proposed Gto2 active site containing Cys46 allows Grx5 to retain some activity against HED. Therefore the residues adjacent to the respective active cysteine residues in Gto2 and Grx5 are important determinants for the thiol transferase activity against small disulphide-containing molecules.     

Potentiometric determination of cysteine with thiol sensitive silver-mercury electrode

A potentiometric procedure for cysteine thiol group concentration monitoring in media generating free radicals was developed using a thiol specific silver-mercury electrode. Electrolytic deposition of mercury on a silver wire and treatment with 20 mM cysteine in 0.5 M NaOH were used to produce the electrode. A silver-chloride electrode in saturated KCl was the reference. A glass capillary with 1 M KNO3 in 1% agarose gel was the liquid junction. The electrode responded to cysteine concentration in the range from 0.01 to 20 mM yielding a perfect linear relationship for the dependence of log [cysteine] versus electrode potential [mV], with b0 (constant) = -373.43 [mV], b1 (slope) = -53.82 and correlation coefficient r2 = 0.97. The electrode potential change per decade of cysteine concentration was 57 mV. The minimal measurable signal response was at a cysteine concentration of 0.01 mM. The signal CV amounted to 4-6% for cysteine concentrations of 0.01 to 0.05 mM and to less than 1% for cysteine concentrations of 0.5 to 20 mM. The response time ranged from about 100 s for cysteine concentrations of 0.01 to 0.1 mM to 30 s at higher cysteine concentrations. The standard curve reproducibility was the best at cysteine concentrations from 0.1 to 20 mM. In a reaction medium containing cysteine and copper(II)-histidine complex ([His-Cu]2+) solution in 55 mM phosphate buffer pH 7.4 the electrode adequately responded to changes in cysteine concentration. Beside cysteine, the silver-mercury electrode responded also to thiol groups of homocysteine and glutathione, however, the Nernst equation slope was about half of that for cysteine.     

Characterization of hemin antibacterial action on Staphylococcus aureus

Abstract The mechanism of inactivation of Staphylococcas aureus cells by hemin is described. Protection experiments by sulfhydryl reagents such as cysteine, mercaptoethanol, glutathione or thioglycolate in their reduced form prevent S. aureus bacteria from inactivation by hemin (1.5 × 10⁻⁵ M). The treatment of bacteria by hemin in the presence of one of those reagents (1 × 10⁻² M) showed that the growth rate and viability of the culture remained unchaged. On the other hand sulfhydryl reagents did not prevent the binding of hemin to the bacteria. When cysteine or glutathione were introduced to a culture after exposure to hemin it could neither reverse the damage done to the cells nor shorten the time of the culture's recovery. Another type of protection was obtained by addition of serum albumin which prevented hemin molecules from binding to the bacterial envelopes. Furthermore, when albumin was introduced after the bacteria were treated by hemin it prevented further damage to the survivors and thus shortened the time required for recovery. None of the singlet oxygen quenchers or hydroxyl radical scavengers could protect the bacteria from hemin inactivation. The mechanism by which hemin affects S. aureus is assumed to be by oxidizing a major system within the cell.     

Covalent binding to glutathione of the DNA-alkylating antitumor agent, S23906-1.

The benzoacronycine derivative, S23906-1, was characterized recently as a novel potent antitumor agent through alkylation of the N2 position of guanines in DNA. We show here that its reactivity towards DNA can be modulated by glutathione (GSH). The formation of covalent adducts between GSH and S23906-1 was evidenced by EI-MS, and the use of different GSH derivatives, amino acids and dipeptides revealed that the cysteine thiol group is absolutely required for complex formation because glutathione disulfide (GSSG) and other S-blocked derivatives failed to react covalently with S23906-1. Gel shift assays and fluorescence measurements indicated that the binding of S23906-1 to DNA and to GSH are mutually exclusive. Binding of S23906-1 to an excess of GSH prevents DNA alkylation. Additional EI-MS measurements performed with the mixed diester, S28053-1, showed that the acetate leaving group at the C1 position is the main reactive site in the drug: a reaction scheme common to GSH and guanines is presented. At the cellular level, the presence of GSH slightly reduces the cytotoxic potential of S23906-1 towards KB-3-1 epidermoid carcinoma cells. The GSH-induced threefold reduction of the cytotoxicity of S23906-1 is attributed to the reduced formation of lethal drug-DNA covalent complexes in cells. Treatment of the cells with buthionine sulfoximine, an inhibitor of GSH biosynthesis, facilitates the formation of drug-DNA adducts and promotes the cytotoxic activity. This study identifies GSH as a reactant for the antitumor drug, S23906-1, and illustrates a pathway by which GSH may modulate the cellular sensitivity to this DNA alkylating agent. The results presented here, using GSH as a biological nucleophile, fully support our initial hypothesis that DNA alkylation is the major mechanism of action of the promising anticancer drug S23906-1.     

Equilibrium characterization of the As(III)-cysteine and the As(III)-glutathione systems in aqueous solution

Some arsenic compounds were the first antimicrobial agents specifically synthesized for the treatment of infectious diseases such as syphilis and trypanosomiasis. More recently, arsenic trioxide has been shown to be efficient in the treatment of acute promyelocytic leukemia. The exact mechanism of action has not been elucidated yet, but it seems to be related to arsenic binding to vicinal thiol groups of regulatory proteins. Glutathione is the major intracellular thiol and plays important roles in the cellular defense and metabolism. This paper reports on a study of the interactions between arsenic(III) and either cysteine or glutathione in aqueous solution. The behavior observed for the As(III)-glutathione system is very similar to that of As(III)-cysteine. In both cases, the formation of two complexes in aqueous solution was evidenced by NMR and electronic spectroscopies and by potentiometry. The formation constants of the cysteine complexes [As(H(-1)Cys)(3)], log K = 29.84(6), and [As(H(-2)Cys)(OH)(2)](-), log K = 12.01(9), and of the glutathione complexes [As(H(-2)GS)(3)](3-), log K = 32.0(6), and [As(H(-3)GS)(OH)(2)](2-), log K = 10(3) were calculated from potentiometric and spectroscopic data. In both cases, the [As(HL)(3)] species, in which the amine groups are protonated, predominate from acidic to neutral media, and the [As(L)(OH)(2)] species appear in basic medium (the charges were omitted for the sake of simplicity). Spectroscopic data clearly show that the arsenite-binding site in both complexes is the sulfur atom of cysteine. In the [As(L)(OH)(2)] species, the coordination sphere is completed by two hydroxyl groups. In both cases, arsenic probably adopts a trigonal pyramidal geometry. Above pH 10, the formation of [As(OH)(2)O](-) excludes the thiolates from arsenic coordination sites. At physiological pH, almost 80% of the ligand is present as [As(HL)(3)].     

Kinetic characterization of native and cysteine 112-modified glutathione S-transferase A1-1: reassessment of nonsubstrate ligand binding

We have prepared human glutathione S-transferase isoform A1-1 (GST A1-1) which has been chemically modified at cysteine 112. These modifications include formation of mixed disulfides with glutathione ("glutathiolation") and cross-linkage of the GST dimer with bis-maleimides reacting with the equivalent Cys 112 residues of the two monomers. This residue (Cys 112) lies adjacent to the hydrophobic substrate binding site, and its side chain thiol projects into the large, solvent-filled cleft which is widely reported in the literature to be the binding site of nonsubstrate ligands. Both types of modification block this intersubunit cleft region and significantly change its chemical environment. Kinetic experiments with these altered enzymes revealed that neither type of modification affects the catalytic activity of GST A1-1 or the binding of nonsubstrate ligands. The lack of an effect on glutathione conjugation activity is somewhat surprising given the proximity of cysteine 112 to the hydrophobic substrate binding site. More surprising, however, is the observation that modification at cysteine 112 has no effect on the binding of nonsubstrate ligands. Furthermore, two of these ligands, lithocholic acid and estradiol disulfate, unexpectedly exhibited competitive inhibition of the unmodified enzyme, suggesting that they bind in the hydrophobic substrate site rather than some accessory ligand binding site. Together, these results strongly argue against the intersubunit cleft as the nonsubstrate ligand binding site and prompt a reassessment of how these ligands interact with GST A1-1.     

Coordination chemistry of glutathione

The metal ion coordination abilities of reduced and oxidized glutathione are reviewed. Reduced glutathione (GSH) is a very versatile ligand, forming stable complexes with both hard and soft metal ions. Several general binding modes of GSH are described. Soft metal ions coordinate exclusively or primarily through thiol sulfur. Hard ones prefer the amino acid-like moiety of the glutamic acid residue. Several transition metal ions can additionally coordinate to the peptide nitrogen of the gamma-Glu-Cys bond. Oxidized glutathione lacks the thiol function. Nevertheless, it proves to be a surprisingly efficient ligand for a range of metal ions, coordinating them primarily through the donors of the glutamic acid residue.     

The specific cleavage of DNA with ultrasound

Cleavages of double-stranded DNA fragments of known base pair sequence upon ultrasound irradiation at 22 and 44 kHz were studied by gel electrophoresis. The cleavage rate is found to be strongly dependent on the DNA fragment length, pH, temperature and ionic strength of the solution under study. The cleavage of double-stranded DNA occurs predominantly at sites containing alternating 5'-CpG-3' sequences. The breakage of phosphatediester bond takes place between C and G in such a way that phosphate group at the 5'-end of the guanine residue remain intact. The cleavage rate at a given DNA site is found to depend on base pair sequences at adjacent sites. Distinctly different cleavage patterns are observed when free DNA and DNA complexes with cys-diammine-Pt-bridged bis-netropsin were irradiated by ultrasound. The observed effect can be attributed to local DNA conformation changes induced upon complex formation between ligand and DNA.