Rapid Equilibrium Kinetic Analysis of Arsenite Methylation Catalyzed by Recombinant Human Arsenic (+3 Oxidation State) Methyltransferase (hAS3MT)

In the human body, arsenic is metabolized by methylation. Understanding this process is important and provides insight into the relationship between arsenic and its related diseases. We used the rapid equilibrium kinetic model to study the reaction sequence of arsenite methylation. The results suggest that the mechanism for arsenite methylation is a completely ordered mechanism that is also of general interest in reaction systems with different reductants, such as tris(2-carboxyethyl)phosphine, cysteine, and glutathione. In the reaction, cysteine residues of recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) coordinate with arsenicals and involve the methyl transfer step. S-Adenosyl-l-methionine (AdoMet) is the first-order reactant, which modulates the conformation of hAS3MT to a best matched state by hydrophobic interaction. As the second-order reactant, reductant reduces the disulfide bond, most likely between Cys-250 and another cysteine residue of hAS3MT, and exposes the active site cysteine residues for binding trivalent inorganic arsenic (iAs³⁺) to give monomethylarsonic dicysteine (MADC³⁺). In addition, the reaction can be extended to further methylate MADC³⁺ to dimethylarsinic cysteine (DAMC³⁺). In the methylation reaction, the β-pleated sheet content of hAS3MT is increased, and the hydrophobicity of the microenvironment around the active sites is decreased. Similarly, we confirm that both the high β-pleated sheet content of hAS3MT and the high dissociation ability of the enzyme-AdoMet-reductant improve the yield of dimethylated arsenicals.     

Platinum(II) binding to metallothioneins

The reaction of equine renal metallothionein (MT) with excess K2PtCl4 at pH 2 results in a polymeric adduct containing 17 +/- 2 mol Pt/mol MT. A monomeric adduct containing 7 mol Pt/mol MT is obtained at neutral pH. Rates of reaction of Pt7MT with DTNB and iodoacetic acid are consistent with Pt2+ to cysteine thiolate coordination, and the extent of reaction in both cases is 11 +/- 2 mol cys/mol MT. Adducts from the reaction of K2PtCl4 with apoMT chemically modified at the N-terminal methionine residue, Cd7MT, and native MT are also reported. A structural model of Pt7MT is proposed in which the square planar tetrathiolate Pt(II) unit is incorporated into a three-metal beta cluster. Implications for the metabolism of platinum anticancer drugs are discussed.     

In vitro activation of soluble guanylyl cyclase and nitric oxide release: a comparison of NO donors and NO mimetics

Nitric oxide (NO) performs a central role in biological systems, binding to the heme site of soluble guanylyl cyclase (sGC), leading to enzyme activation and elevation of intracellular levels of cGMP. Organic nitrates, in particular, nitroglycerin (GTN), are clinically important nitrovasodilators that function as NO-mimetics in biological systems. Comparison of sGC activation data with electrochemically measured rates of NO release for genuine NO donors, NONOates and nitrosothiols, yields an excellent correlation between the EC(50) for sGC activation and the rate constant for NO release, k(NO). However, activation of sGC by GTN and the nitrates has very different characteristics, including the requirement for specific added thiols, for example, cysteine. The reaction of GTN with cysteine in anaerobic solution yields NO slowly, and NO release, measured by chemiluminescence detection, is quenched by added metal ion chelator. The generation of NO under aerobic conditions is 100-fold slower than the anaerobic reaction. Furthermore, NO release from the reaction of GTN with cysteine in phosphate buffer is too slow to account for sGC activation by GTN/cysteine. The slow rate of the chemical reaction to release NO suggests that nitrates can activate sGC by an NO-independent mechanism. In contrast to the genuine NO donors, GTN behaves as a partial agonist with respect to sGC activation, but in the presence of the allosteric sGC activator, YC-1, GTN exhibits full agonist activity.     

Rates of metallothionein synthesis, degradation and accretion in a chicken macrophage cell line

To understand the regulation of metallothionein (MT) accretion in a chicken-macrophage cell line, fractional rates of MT synthesis (FRS) and degradation (FRD) were measured by following decay kinetics of [35S]cysteine in MT. To obtain valid measurements, we added various amounts of cysteine to medium to ensure that the isotope tracer was adequately diluted after MT was labeled in the presence of various levels of zinc. We also demonstrated that the measured fractional rate of MT accretion closely approximated the difference between FRS and FRD. All fractional rates were similar for the two MT isoforms isolated. FRD did not change during the 27-hr decay period, but FRS and fractional rate of MT accretion changed over time. FRS of MT was 0.097 and 0.012 hr-1 from 0 to 9 and 9 to 27 hr, respectively, after cells were incubated in medium supplemented with 50 microM zinc. FRD of MT was 0.020 hr-1. Addition of 1100 microM unlabeled cysteine to medium supplemented with 50 microM zinc increased FRD and decreased FRS and fractional rate of MT accretion, as compared with not adding cysteine. Overall, these results indicate that rates of MT synthesis and degradation can both regulate MT accretion. Further experiments with various amounts of zinc and cysteine added to medium suggested that the effect of added cysteine on MT fractional rates was due to chelation of unbound zinc. Elimination of the cysteine effect on MT fractional rates was accomplished by adding more zinc to cysteine-supplemented medium. Thus, the concentration of unbound zinc affects the rates of MT synthesis and degradation.     

Study of the redox properties of metallothionein in vitro by reacting with DsbA protein

Mammalian metallothionein (MT) contains 20 cysteine residues involved in the two metal clusters without a disulfide bond. The redox reaction of the Cys thiols was proposed to be associated with the metal distribution of MT. The E. coli DsbA protein is extremely active in facilitating thiol/disulfide exchange both in vivo and in vitro. To further investigate the redox properties of MT, reaction between MT and DsbA was carried out in vitro by fluorescence detection. Equilibrium characterization indicates that the reaction is stoichiometric (1:1) under certain conditions. Kinetic study gives a rate constant of the redox reaction of 4.42 × 10⁵ sec^–1 M^–1, which is 10³-fold larger than that of glutathione reacting with DsbA. Metal-free MT (apo-MT) shows a higher equilibrium reduction potential than MT, but exhibits an indistinguishable kinetic rate. Oxidation of MT by DsbA leads to metal release from the clusters. The characteristic fluorescence increase during reduction of DsbA may provide a sensitive probe for exploring the redox properties of some reductants of biological interest. The result also implies that oxidation of Cys thiols may influence the metal release or delivery from MT.     

Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates

ABSTRACT: BACKGROUND: While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction. Organic solvents, including DMSO and formamide, have been often employed as additives to increase the efficiency of amplification of high GC content (GC > 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors such as phenolic compounds. FINDINGS: Significant increase in PCR amplification yields of GC-rich DNA targets ranging in sizes from 0.4 kb to 7.1 kb were achieved by using BSA as a co-additive along with DMSO and formamide. Notably, enhancing effects of BSA occurs in the initial PCR cycles with BSA additions having no detrimental impact on PCR yield or specificity. When a PCR was set up such that the cycling parameters paused after every ten cycles to allow for supplementation of BSA, combining BSA and organic solvent produced significantly higher yields relative to conditions using the solvent alone. The co-enhancing effects of BSA in presence of organic solvents were also obtained in other PCR applications, including site-directed mutagenesis and overlap extension PCR. CONCLUSIONS: BSA significantly enhances PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to amplify. The strategy outlined here provides a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range.     

Mitral tetrahedron as a geometrical surrogate for chronic ischemic mitral regurgitation

The present study tests the hypothesis that a mitral tetrahedron (MT) is a useful geometrical surrogate for assessment of chronic ischemic mitral regurgitation (CIMR). Fifty-eight subjects were divided into three groups on the basis of left ventricular ejection fraction (LVEF) and the presence or absence of CIMR: LVEF > or =0.5 and negative CIMR (group 1, n = 28), LVEF <0.5 and negative CIMR (group 2, n = 12), and LVEF <0.5 and positive CIMR (group 3, n = 18). MT was defined by its four vertices at the anterior annulus, posterior annulus, and medial and lateral papillary muscle roots, determined by MRI at peak systole. The results showed no clear cutoff values of MT parameters between groups 2 and 1. In contrast, all MT indexes were significantly different between groups 3 and 2 (P < 0.05), and significant cutoff values differentiated the two groups. A scoring system employing parameters of the whole MT confirmed the absence of CIMR with total edge length index <268 mm/BSA(1/3), total surface area index <2,528 mm(2)/BSA(2/3), and volume index <5,089 mm(3)/BSA (where BSA is body surface area). The sensitivity, specificity, and positive and negative predictive values were 1.00. This preliminary study demonstrates that MT might serve as a good geometrical surrogate for assessing CIMR. The derived geometrical criteria of MT may be useful in surgical correction of CIMR.     

Cone packing model — a geometrical approach to coordination and organometallic chemistry of lanthanides and actinides

By projection of the ligands to a unit sphere centred at the metal ion, steric effects, both local and overall, can be studied quantitatively in terms of solid angle factor (SAF), fan angle (FA), coordination vector, gap and hole. The packing saturation rule and the packing centre rule have been determined by treatment of more than 400 structures of lanthanide and actinide coordination and organometallic compounds. A ligand packing model was thus adopted with cones joined at the same apex.‘Coordination Saturation’ in f-block chemistry was clarified to be saturation in coordination space, implying an equilibrium between bonding and steric effects. Molecular structures could be simulated on the basis of a uniform packing principle. The cone packing model has been applied to inorganic preparation, synergistic extraction and structure prediction. These predictions have been clearly substantiated experimentally, and the results are reported.A dynamic packing model was also suggested to study the reaction pathways by simulating the relative positions during the reaction process and the size and shape of holes, which correspond to the reaction areas in the coordination sphere. Lewis base association and dissociation, and the metal- carbon bond thermolysis of structural pattern MCp₃L were explained as examples.     

Effects of heparin, sperm concentration and bull variation on in vitro fertilization of bovine oocytes in a protein-free medium

The present study was conducted to examine the effects of heparin, sperm concentration and bull variation on the fertilization of bovine oocytes in a protein-free medium supplemented with polyvinyl alcohol and subsequent in vitro development of fertilized embryos. The effects in protein-free medium were compared with those in medium supplemented with bovine serum albumin (BSA). In the presence of heparin (1, 10 and 100 microg/ml), nearly all the oocytes were fertilized with and without BSA. In the absence of BSA, polyspermy was lower (4 to 15%) than in its presence (15 to 48%; P < 0.05). An increase in sperm concentration from 1 x 10(4) cells/ml during insemination enhanced fertilization rate up to 1 x 10(6) cells/ml with and without BSA (14 to 90% and 3 to 77%, respectively). In the absence of BSA, the highest concentration of spermatozoa (1 x 10(7) cells/ml) gave a lower fertilization rate (55%) than that at 1 x 10(6) cells/ml (77%; P < 0.05). Polyspermy neither increased nor decreased sperm concentration without BSA (0 to 8%; P > 0.05). The effects of spermatozoa from 5 different bulls chosen randomly on in vitro fertilization in medium without BSA were examined. Individual bull variation in fertilization rate (36 to 95%) was noted at 3 different heparin concentrations (1, 10 and 100 microg/ml). Polyspermic fertilization was low (0 to 14%) and was the same for all bulls at all heparin concentrations. Embryos fertilized without BSA developed to the blastocyst stage at the same rate (27%) as those with BSA (33%; P > 0.05).     

Technetium-binding in labelled HYNIC-peptide conjugates: Role of coordinating amino acids

Electrospray mass spectrometry (ESMS) of certain peptides labelled with ^99mTc via hydrazinonicotinamide (HYNIC) with tricine as co-ligand shows one Tc-bound tricine, whereas typically two are observed. We speculated that this was due to coordination of a neighbouring histidine (His) or glutamate (Glu). To investigate this possibility, several short peptides incorporating lysine (HYNIC), with and without His and Glu at different positions in the sequence, were radiolabelled with ^99mTc, using tricine, ethylenediaminediacetic acid (EDDA) and nicotinic acid as co-ligands. The products were examined by HPLC-ESMS, cysteine challenge and bovine serum albumin (BSA) challenge. Peptides with His nearby on either side of lysine (HYNIC) contained only one tricine and showed markedly enhanced structural homogeneity and stability to cysteine challenge and BSA binding, except those with His located at the N-terminus. Peptides without His, or with neighbouring N-terminal His, contained two tricines and were less stable to cysteine challenge and BSA binding. Glu participated in Tc-binding but did not enhance stability. We conclude that neighbouring His or Glu side chains coordinate to Tc and this could alter peptide or protein conformation. Inclusion of His in a neighbouring position to lysine (HYNIC) enhances stability, improves homogeneity and reduces the demand of the metal center for binding to additional co-ligands.     

Glutathione status in constituted physiological fluids containing albumin

1. Glutathione (GSH) and cysteine, added to the constituted incubation medium, rapidly disappeared from the medium in the presence of bovine serum albumin (BSA). The major portions of added GSH and cysteine were oxidized. Only a fraction was recovered as cysteine-GSH mixed disulfide in case of GSH. About 15-30% cysteine or GSH were not recovered in the media. 2. The rate of GSH oxidation was linear with time, however, GSH disappearance was not linear with GSH concentrations. 3. Oxidation of GSH to GSSG in the albumin supplemented media was greater under O2 atmosphere, but was significantly decreased under N2 atmosphere. 4. Catalase, a peroxy radical scavenger, but not dimethyl pyroline N-oxide (DMPO), N-tertbutyl-2(-2 sulfophenyl)-nitrone (NTBSPN), mannitol or superoxide dismutase (SOD), decreased BSA mediated GSH oxidation. 5. GSH oxidation was abolished when mono- or divalent metal ions were absent in the BSA supplemented media. 6. Alkaline pH favored and acidic pH inhibited GSH oxidation. GSH oxidation was maximum above pH 7.4. GSH oxidation was minimal in the media containing boiled BSA. 7. A reaction mechanism involving the mixed GSH-BSA disulfide formation, followed by the reduction of these disulfides by GSH and subsequent release of GSSG is proposed.     

Electron paramagnetic resonance study of nitrosylprotoheme dimethyl ester complexes with aliphatic nitrogenous bases: characterization of the axial ligand trans to the nitrosyl group in nitrosylhemoproteins

The interaction of nitrosyl(protoporphyrin IX dimethyl ester) iron (II) (Fe(PPDME)(NO)) with aliphatic amines, anilines, and cyclic imines has been studied by electron paramagnetic resonance (EPR) measurements at room temperature and at 77K. At room temperature, the bases studied here were divided into two groups according to the exchange rate between two EPR-positive species, Fe(PPDME)(NO) and Fe(PPDME)(NO)B, in equilibrium; Fe(PPDME)(NO) + B

Fe(PPDME)-(NO)B. The Fe(PPDME)(NO)-base system with a fast exchange rate on the EPR time scale had a smaller equilibrium constant (K) than that with a slow rate. The EPR spectra of the Fe(PPDME)(NO)-base system both at room temperature and at 77K were markedly influenced by the steric interaction of the base with the porphyrin core.     

Fourier transform IR attenuated total reflectance spectroscopy studies of cysteine-induced changes in secondary conformations of bovine serum albumin after UV-B irradiation

Fourier transform IR spectroscopy equipped with attenuated total reflection was used to investigate the cysteine-induced alteration of the protein secondary structure of bovine serum albumin (BSA) in aqueous solution before and after UV-B irradiation. Several amino acids were also studied. The results indicate the unchanged IR spectra of BSA coincubated with amino acids, except cysteine, did not change after 72-h UV-B irradiation. There was no difference in the IR spectrum of the unirradiated BSA coincubated with cysteine. A shoulder at 1620 cm(-1) attributed to the intermolecular beta-sheet structure was observed for the IR spectrum of BSA coincubated with cysteine after 72-h UV-B irradiation. Moreover, the peak intensity at 1303 cm(-1) that is due the alpha-helix structure was reduced, but the peak intensity at 1247 cm(-1) corresponding to beta-sheet structures was increased. Longer UV-B exposure for a BSA solution coincubated with cysteine changed the BSA solution from clear to viscous to gel form in which a transparent gel and another white gel were simultaneously observed. A gradual IR spectral alteration was found for BSA coincubated with cysteine and subjected to increased UV-B irradiation. The longer UV-B irradiation yielded increased intensity at 1620 cm(-1). The second-derivative IR peaks at 1655, 1631, and 1548 cm(-1) were shifted to 1650, 1620, and 1544 cm(-1), respectively, by the increase of UV-B irradiation, suggesting a progressive transformation from an alpha-helix to an intermolecular beta-sheet structure for BSA coincubated with cysteine. This strongly implies that longer UV-B exposure time for the BSA solution in the presence of cysteine did alter the protein secondary structures of BSA more, thus inducing gel formation by protein aggregation.     

In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium

The effect of BSA, caffeine and calcium was studied on the penetration of pig oocytes by frozen-thawed spermatozoa in a modified Tris-buffered medium (mTBM) without added bicarbonate. Pig cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. The COC were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with spermatozoa for 6 h in mTBM containing caffeine (5 mM) and 0.1 or 0.4% BSA (Experiment 1). In Experiment 2, oocytes were inseminated in mTBM containing 0.1% BSA and various concentrations of caffeine (0 to 5 mM). In Experiment 3, insemination was carried out in mTBM containing 0.1% BSA, 1 mM caffeine and various concentrations of Ca(2+) (0.5 to 10 mM). Supplementation of mTBM with either 0.1 or 0.4% BSA resulted a high penetration rate with a high polyspermy rate. However, the mean number of spermatozoa per oocyte was significantly higher at 0.4% than at 0.1% BSA. The penetration rate, polyspermy rate and mean number of spermatozoa per oocyte were all significantly higher when 1 to 5 mM caffeine were added to the medium than in caffeine-free medium. No penetration was observed in the presence of 0.5 mM Ca(2+). The penetration rate was significantly increased from 12 to 92% at 2.5 to 10 mM Ca(2+). The mean number of spermatozoa per oocyte did not differ between 2.5 and 5 mM Ca(2+) but increased significantly at 7.5 and 10 mM. These results show the successful in vitro penetration of pig oocytes in a chemically semidefined medium without added bicarbonate. Although BSA and caffeine can modulate the rate of sperm penetration, calcium seems to be an important regulatory ion.     

Solution structure of Cu6 metallothionein from the fungus Neurospora crassa.

The 3D-solution structure of Neurospora crassa Cu(6)-metallothionein (NcMT) polypeptide backbone was determined using homonuclear, multidimensional (1)H-NMR spectroscopy. It represents a new metallothionein (MT) fold with a protein chain where the N-terminal half is left-handed and the C-terminal half right-handedly folded around a copper(I)-sulfur cluster. As seen with other MTs, the protein lacks definable secondary structural elements; however, the polypeptide fold is unique. The metal coordination and the cysteine spacing defines this unique fold. NcMT is only the second MT in the copper-bound form to be structurally characterized and the first containing the -CxCxxxxxCxC- motif. This motif is found in a variety of mammalian MTs and metalloregulatory proteins. The in vitro formation of the Cu(6)NcMT identical to the native Cu(6)NcMT was dependent upon the prior formation of the Zn(3)NcMT and its titration with Cu(I). The enhanced sensitivity and resolution of the 800 MHz (1)H-NMR spectral data permitted the 3D structure determination of the polypeptide backbone without the substitution and utilization of the NMR active spin 1/2 metals such as (113)Cd and (109)Ag. These restraints have been necessary to establish specific metal to cysteine restraints in 3D structural studies on this family of proteins when using lower field, less sensitive (1)H-NMR spectral data. The accuracy of the structure calculated without these constraints is, however, supported by the similarities of the 800 MHz structures of the alpha-domain of mouse MT1 compared to the one recalculated without metal-cysteine connectivities.     

Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase

The reactivity of simple alkyl thiolates with N-ethylmaleimide (NEM) follows the Br?nsted equation, log kS- = log G + beta pK, with G = 790 M-1 min-1 and beta = 0.43. The rate constant for the reaction of the thiolate of 2-mercaptoethanol with NEM is 10(7) M-1 min-1, whereas the rate constant for the reaction of the protonated thiol is less than 0.0002 M-1 min-1. The intrinsic reactivity of the protonated thiol (SH) is over (5 X 10(10]-fold less than the thiolate (S-) and makes a negligible contribution to the reactivity of thiols toward NEM. The rate of NEM modification of chalcone isomerase was conveniently measured by following the concomitant loss in enzymatic activity. The pseudo-first-order rate constants for inactivation show a linear dependence on the concentration of NEM up to 200 mM and yield no evidence for noncovalent binding of NEM to the enzyme. Evidence is presented demonstrating that the modification of chalcone isomerase by NEM is limited to a single cysteine residue over a wide range of pH. Kinetic protection against inactivation and modification by NEM is provided by competitive inhibitors and supports the assignment of this cysteine residue to be at or near the active site of chalcone isomerase. The pH dependence of inactivation of the enzyme by NEM indicates a pK of 9.2 for the cysteine residue in chalcone isomerase. At high pH, the enzymatic thiolate is only (3 X 10(-5))-fold as reactive as a low molecular weight alkyl thiolate of the same pK, suggesting a large steric inhibition of reaction on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrative roles of nitric oxide and hydrogen sulfide in melatonin-induced tolerance of pepper (Capsicum annuum L.) plants to iron deficiency and salt stress alone or in combination

There seems to be no report in the literature on the effect of melatonin (MT) in relieving the detrimental effects of combined application of salt stress (SS) and iron deficiency (ID). Therefore, the effect of MT on the accumulation/synthesis of endogenous nitric oxide (NO) and hydrogen sulphide (H₂S) and how far these molecules are involved in MT-improved tolerance to the combined application of ID and SS in pepper (Capsicum annuum L) were tested. Hence, two individual trials were set up. The treatments in the first experiment comprised: Control, ID (0.1 mM FeSO₄), SS (100 mM NaCl) and ID + SS. The detrimental effects of combined stresses were more prominent than those by either of the single stress, with respect to growth, oxidative stress and antioxidant defense attributes. Single stress or both in combination improved the endogenous H₂S and NO, and foliar-applied MT (100 µM) led to a further increase in NO and H₂S levels. In the second experiment, 0.1 mM scavenger of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and that of H₂S, hypotuarine (HT) were applied along with MT to get further evidence whether NO and H₂S are involved in MT-induced tolerance to ID and SS. MT combined with cPTIO and HT under a single or combined stress showed that NO effect was reversed by the NO scavenger, cPTIO, alone but the H₂S effect was inhibited by both scavengers. These findings suggested that tolerance to ID and SS induced by MT may be involved in downstream signal crosstalk between NO and H₂S.     

Protective effect of metallothionein to ras DNA damage induced by hydrogen peroxide and ferric ion-nitrilotriacetic acid

Metallothionein (MT) is a strong antioxidant, due to a large number of thiol groups in the MT molecule and MT has been found in the nucleus. To investigate whether MT can directly protect DNA from damage induced by hydroxyl radical, the effects of MTs on DNA strand scission due to incubation with ferric ion-nitrilotriacetic acid and H2O2 (Fe3+ -NTA/H2O2) were studied. The Fe3+-NTA/H2O2 resulted in a higher rate of deoxyribose degradation, compared to incubation of Fe3+/H2O2, presumably mediated by the formation of hydroxyl radicals (*OH). This degradation was inhibited by either Zn-MT or Cd-MT, but not by Zn2+ or Cd2+ at similar concentrations. The Fe3+ -NTA/H2O2 resulted in a concentration dependent of increase in DNA strand scission. Damage to the sugar-phosphodiester chain was predominant over chemical modifications of the base moieties. Incubation with either Zn-MT or Cd-MT inhibited DNA damage by approximately 50%. Preincubation of MT with EDTA and N-ethylmaleimide, to alkylate sulfhydryl groups of MT, resulted in MT that was no longer able to inhibit DNA damage. These results indicates that MT can protect DNA from hydroxyl radical attack and that the cysteine thiol groups of MT may be involved in its nuclear antioxidant properties.     

Nitric oxide scavenging by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated ascorbate reduction of methemoglobin

NADH-dependent NO scavenging in barley extracts is linked to hemoglobin (Hb) expression and is inhibited by SH-reagents. Barley Hb has a single cysteine residue. To determine whether this cysteine was critical for NO scavenging, barley Hb and a mutated version, in which the single Cys⁷⁹ was replaced by Ser, were over-expressed in Escherichia coli and purified to near homogeneity. The purified proteins exhibited very low NO-scavenging activity (12–14 nmol min⁻¹ mg⁻¹ protein) in the presence of NADH or NADPH. This activity was insensitive to SH-reagents. Addition of an extract from barley roots to either of the purified proteins resulted in high NADH-dependent NO turnover in a reaction that was sensitive to SH-reagents. A protein was purified from barley roots and identified by mass-spectrometry analysis as a cytosolic monodehydroascorbate reductase. It efficiently supported NADH-dependent NO scavenging in the presence of either native or mutated barley Hb. Ascorbate strongly facilitated the rate of metHb reduction. The K _m for Hb was 0.3 μM, for ascorbate 0.6 mM and for NADH 4 μM. The reaction in the presence of monodehydroascorbate reductase was sensitive to SH-reagents with either form of the Hb. We conclude that metHb reduction and NO turnover do not involve direct participation of the Cys⁷⁹ residue of barley Hb. NO scavenging is facilitated by monodehydroascorbate reductase mediating a coupled reaction involving ferric Hb reduction in the presence of ascorbate and NADH.