OH-radical-type reactive oxygen species derived from superoxide and nitric oxide: a sensitive method for their determination and differentiation

Reactive oxygen species are involved in many diseases where the radical species OH, peroxynitrite and the non-radical, hypochlorous acid, play an outstanding role. The formation of OH-type oxidants is essentially confined to a few types of reactions. The most prominent ones are the one-electron reduction of hydrogen peroxide by F2+ or Cu+ -ions (Fenton-type reactions), reaction of hypochlorite with superoxide and finally formation and decay of peroxynitrite (ONOOH), formed from superoxide and NO. In this communication we wish to report on a simple model system allowing to differentiate between these ROS: ethene formation from ACC is only detectable in the presence of hypochlorite (v. Kruedener et al, 1995) and not detectable with Fenton-type oxidants or SIN-1 (3-morpholinosydonimine, a peroxynitrite generator by releasing sequentially superoxide and NO) at 10 microM concentrations. On the other hand, ethene formation from KMB is negligible in the presence of hypochlorite but proceeds rapidly with Fenton-type oxidants (4 microM H2O2; 4 microM Fe2+) as well as with 1 microM SIN-1. Stimulation of Fenton-type oxidants and not of SIN-1 by EDTA and characteristic patterns of inhibition by SOD, catalases, hemoglobin and uric acid allow a differentiation between these two potential precursors of OH-radicals. Synthetic ONOOH shows different reaction kinetics as compared to SIN-1. Inhibition of ONOOH-dependent ethene formation by different compounds occurs more or less "random" indicating an unspecific influence of proteins and also small molecules. Comparison of the individual inhibition types of several selected compounds allows a differential analysis as to the generation pathway of the final oxidants, OH- radical or peroxynitrite.     

Kinetics of the competitive degradation of deoxyribose and other biomolecules by hydroxyl radicals produced by the Fenton reaction

The objective of this work is to reexamine the competitive degradation of deoxyribose by hydroxyl radicals (.OH) produced by the reaction between H2O2 and Fe(2+)-EDTA. The .OH radicals produced attack deoxyribose (D, rate constant kD) and eventually an .OH scavenger (S, rate constant kS). First, we examined the effect of [D], [H2O2], [Fe(2+)-EDTA], [EDTA]/[Fe2+] ratio and reaction time on the rate of D degradation, measured as the absorbance of the chromogen formed between the product of the reaction D + .OH (malondialdehyde) and thiobarbituric acid. In particular, it was showed that under our experimental conditions ([D] = 3 mM, [H2O2] = 0.85 mM, [Fe2+] = 0.13 mM), the rate of overall process is first order in Fe2+, zero order in H2O2 and is maximal for a ratio [EDTA]/[Fe2+] = 1.1. Second, the kinetics of .OH radical reaction in competition experiments between D and S (mannitol) was investigated. The results show that the ratio of the rates of D degradation in the absence (VD) and in the presence (VDS) of S should be represented by VD/VDS = 1 + ks[S]/(kD[D] + kx) where kx accounts for the rate of .OH reactions with other reagents such as Fe(2+)-EDTA, H2O2 etc . . . After having determined kx for each set of experimental conditions, we obtained the values of kS/kD by determining the variations of VD/VDS as a function of [S] and [D]. By taking kD = 1.9 x 10(9) M-1s-1 a value of kS = 1.9 x 10(9) M-1s-1 was obtained, very close to that obtained by pulse radiolysis. Finally, the validity of the established relation was confirmed for other biomolecules (methionine, k = 5.6 x 10(9)M-1s-1 and alanine, k = 3.3 x 10(8) M-1s-1). By contrast, it was not applicable to cysteine, thiourea and mercaptoethanol which was attributed to an interaction of the latter scavengers with Fe2+ and/or H2O2.     

Differential effects of DNA supercoiling on radical-mediated DNA strand breaks

Supercoiling is an important feature of DNA physiology in vivo. Given the possibility that the reaction of genotoxic molecules with DNA is affected by the alterations in DNA structure and dynamics that accompany superhelical tension, we have investigated the effect of torsional tension on DNA damage produced by five oxidizing agents: gamma-radiation, peroxynitrite, Fe2+/ EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. With positively supercoiled plasmid DNA prepared by a recently developed technique, we compared the quantity of strand breaks produced by the five agents in negatively and positively supercoiled pUC19. It was observed that strand breaks produced by gamma-radiation, peroxynitrite, and Fe2+/EDTA/H2O2 were insensitive to DNA superhelical tension. These results are consistent with a model in which chemicals that generate highly reactive intermediates (e.g., hydroxyl radical), but do not interact directly with DNA, will be relatively insensitive to the changes in DNA structure and dynamics caused by superhelical tension. In the case of Fe2+ and Cu2+, metals that bind to DNA, only Cu2+/H2O2 proved to be sensitive to DNA superhelical tension. Strand breaks produced by Cu2+/H2O2 in the positively supercoiled substrate occurred at lower Cu concentrations than in negatively supercoiled DNA. Furthermore, a sigmoidal Cu2+/H2O2 damage response was observed in the negatively supercoiled substrate but not in positively supercoiled DNA. The results with Cu2+ suggest that the redox activity, DNA binding orientation, or DNA binding affinity of Cu1+ or Cu2+ is sensitive to superhelical tension, while the results with the other oxidizing agents warrant further investigation into the role of supercoiling in base damage.     

Fe2+, Fe3+, and oxygen react with DNA-derived radicals formed during iron-mediated Fenton reactions

Oxidative DNA damage is decreased by the presence of O2 during Fe(2+)-mediated Fenton reactions when H2O2 is in excess. During these reactions, the presence of DNA increases H2O2 consumption relative to Fe2+ consumption under anaerobic conditions, but decreases H2O2 consumption relative to Fe2+ consumption under aerobic conditions. The pseudobimolecular rate constant of H2O2 consumption is the same under both conditions, however, indicating that the presence of DNA affects the oxidation and/or reduction of the iron pool. To understand the basis of these effects, DNA was replaced with ethanol as a model compound. Computer simulations of Fe2+ and H2O2 consumption were experimentally verified and allowed identification of the predominant reactions leading to the changes in stoichiometry. Based upon these results and upon qualitative and quantitative differences in DNA damages between aerobic and anaerobic conditions, it was concluded that, in the presence of DNA, Fe3+ is reduced by some DNA radicals. However, if O2 is present, these radicals react instead with O2 and the product of these reactions can then oxidize Fe2+. Mechanisms proposed for the alteration by O2 of products from dC- and dG-containing substrates after exposure to Fe and H2O2 fit these general schemes. These results provide another distinction between DNA damage caused by ionizing radiation and that caused by Fenton reactions.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-1-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (.OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the .OH generation. UV and O2 consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H2O2 did not generate any significant amount of .OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H2O2-->Co(II) + .OH + OH-] seems responsible for .OH generation. H2O2 is produced from O2.- via dismutation, O2.- is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or beta-ananyl-3-methyl-L-histidine alters, its oxidation-reduction potential and makes Co(II) capable of generating .OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H2O2-->Co(III) + .OH + OH-]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and beta-ananyl-3-methyl-L-histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Differential effects of diazepam on anxiety in streptozotocin induced diabetic and non-diabetic rats

In the absence of added Fe2+, the ATPase activity of isolated Schizosaccharomyces pombe plasma membranes (5-7 mumol P(i) per mg protein per min) is moderately inhibited by H2O2 in a concentration-dependent manner. Sizable inactivation occurs only at 50-80 mmol/L H2O2. The process, probably a direct oxidative action of H2O2 on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both the K(m) of the enzyme for ATP and the V of ATP splitting. On exposing the membranes to the Fenton reagent (50 mumol/L Fe2+ + 20 mmol/L H2O2), which causes a fast production of HO. radicals, the ATPase is 50-60% inactivated and 90% of added Fe2+ is oxidized to Fe3+ within 1 min. The inactivation occurs only when Fe2+ is added before H2O2 and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO. radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine implies that the process requires the presence of Fe2+ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe2+ for H2O2, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that gives rise to delayed HO. production.     

Oxidative inactivation of brain alkaline phosphatase responsible for hydrolysis of phosphocholine

Alkaline phosphatase, one of the enzymes responsible for the conversion of phosphocholine into choline, was purified from bovine brain membrane, where the phosphatase is bound as glycosylphosphatidylinositol-linked protein, and subjected to oxidative inactivation. The phosphatase activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased slightly after the exposure to H2O2. Inclusion of Cu2+ in the incubation with 1 mM H2O2 led to a rapid decrease of activity in a time- and concentration-dependent manner. In comparison, the H2O2/Cu2+ system was much more effective than the H2O2/Fe2+ system in inactivating brain phosphatase. In a further study, it was observed that the hydroxy radical scavengers mannitol, ethanol, or benzoate failed to prevent against H2O2/Cu2+-induced inactivation of the phosphatase, excluding the involvement of extraneous hydroxy radicals in metal-catalyzed oxidation. In addition, it was found that both substrates, p-nitrophenyl phosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although incomplete, protection against the inactivating action of H2O2/Cu2+. A similar protection was also expressed by divalent metal ions such as Mg2+ or Mn2+. Separately, it was found that H2O2/Fe2+-induced inactivation was prevented by p-nitrophenyl phosphate or Mg2+ but not phosphate ions. Thus, it is implied that phosphocholine-hydrolyzing alkaline phosphatase in brain membrane might be one of enzymes susceptible to metal-catalyzed oxidation.     

Formation of 8-hydroxydeoxyguanosine from 2'-deoxyguanosine and its decomposition by active oxygen

8-hydroxydeoxyguanosine (8-OHdG) was formed from dG and isolated DNA by photosensitization with rose bengal (RB) and methylene blue (MB). 8-OHdG formed from dG was decomposed by the photosensitization with these dyes. Singlet oxygen was concerned with the formation and decomposition of 8-OHdG by photosensitization. Fe++ oxidized dG to 8-OHdG, which was decomposed by the addition of H2O2. 8-OHdG was formed and decomposed by the treatment of dG with Fe++, EDTA and ascorbic acid. Hydroxy radical (.OH) participated in the formation and decomposition of 8-OHdG by Fe++.     

The origin of the hydroxyl radical oxygen in the Fenton reaction

There is an ongoing discussion in the chemical literature regarding the nature of the highly reactive hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction). However, the fundamental experiment of directly determining the source of the hydroxyl radicals formed in the reaction has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with 17O, together with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. ESR experiments were run in phosphate buffer with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, and either H2O2 or H2O labeled with 17O. The hydroxyl radical was generated by addition of Fe2+ ion to H2O2, or as a control, by photolysis of H2O2 in the ESR cavity. Observed ESR spectra were the sum of DMPO/.16OH and DMPO/.17OH radical adduct spectra. Within experimental accuracy, the percentage of 17O-labeled hydroxyl radical trapped by the DMPO was the same as in the original hydrogen peroxide, for either method of hydroxyl radical generation, indicating that the trapped hydroxyl radical was derived exclusively from hydrogen peroxide and that there was no exchange of oxygen atoms between H2O2 and solvent water. Likewise, the complementary reaction with ordinary H2O2 and 17O-labeled water also showed that none of the hydroxyl radical was derived from water. Our results do not preclude the ferryl intermediate, [Fe = O]2+ reacting with DMPO to form DMPO/.OH if the ferryl oxygen is derived from H2O2 rather than from a water ligand.     

Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase AT submicromolar iron concentrations: evidence for metal-catalyzed oxidation

The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe2+ + H2O2 --> HO. + HO- + Fe3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca2+-ATPase by oxygen reactive species at Fe2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe2+, H2O2, and ascorbate. Measuring the rate constants of inactivation (k inact) at different Fe2+ concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 microM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 microM Fe2+. Inhibition was not affected by addition of 200 microM Ca2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu2+ was used instead of Fe2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.     

Initiation of radicals in biochemical systems: ferritin-hydroperoxides-aromatic amines

The rates of initiation of free radicals were determined in systems containing horse spleen ferritin, H2O2, tert-butyl hydroperoxide (TBHP) or cumene hydroperoxide (CHP) in acetate buffer (pH 4.2) or phosphate buffer (pH 6.0) with 10-15% dimethylformamide (DMF). Benzidine (BD), benzidine sulfate (BDS), o-tolidine (TL), 3,3',5,5'-tetramethylbenzidine (TMB), and o-phenylenediamine (PDA) were used as acceptors. In the systems ferritin-H2O2 oxidation of amine acceptor follows Michaelis-Menten kinetics. For all aromatic amines kcat, Km, and their ratios were determined. Peroxidase efficiency of ferritin in the TMB oxidation by hydrogen peroxide (kcat/km) is characterized by a value 2.82.10(3) M-1.sec-1 comparable with ferroxidase efficiency of apoferritin in the oxidation of Fe2+ by oxygen. Reactivity of aromatic amines in the system ferritin-H2O2 is similar to the reactivity registered in their peroxides oxidation and is maximal for TMB and PDA. Bimolecular rate constants of TMB and PDA oxidation in the reaction with H2O2, TBHP, and CHP were compared in acetate buffer (pH 4.2 using 0.5 microM ferritin and 5 mM concentration of each substrate. On the oxidation of both amines activity of oxidants decreased in the following order: H2O2 > TBHP > CHP. A scheme of radical initiation in the systems ferritin-ROOH (H2O2)-amines and the influence of radical acceptors apoferritin and organic co-solvent on the rate of reactions are discussed.     

Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species

Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.     

硫氰酸盐法从含铜黄铁矿焙砂中浸金研究

对用Ｆｅ３＋作氧化剂，ＳＣＮ－作络合剂，从含铜黄铁矿焙砂中提取黄金的工艺条件进行了研究。理论分析和实验结果都证明了该工艺在技术上是可行的。利用了黄铁矿焙砂中的Ｆｅ２Ｏ３经酸分解后而产生的Ｆｅ３＋作氧化剂，可不必再加其它的氧化剂，同时脱铜后又可降低硫氰酸铵的耗量。本工艺还具有设备简单、毒性小、周期短、综合利用程度高等优点。     

Nuclear DNA content and morphological characteristics in the prognosis of adrenocortical carcinoma

Bathophenanthrolinedisulfonic acid (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid [BPS]) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine), chelators of ferrous iron, are often used to determine iron(II) concentrations in various samples and for identifying or measuring iron reduction in biological systems. In this study, the efficacy of ferrozine and BPS to chelate iron(II) reduced from Fe(3+)-ligands by selected reducing agents was determined. Our results indicate that (i) BPS and ferrozine are not equivalent as kinetic indicators of iron reducing activity; (ii) apparent initial rates of reduction of Fe(3+)-ligands by dithiothreitol, as indicated by formation of complexes of iron(II) with either BPS or ferrozine, differed by a factor of 50; and (iii) nonspecific reduction of some Fe(3+)-ligands by both BPS and ferrozine occurred. Under identical conditions, rates of formation of Fe(2+)-ferrozine generally were slower than rates of formation of Fe(2+)-BPS. These data suggest careful consideration should be given in the design of any experiments where kinetics of iron reduction are monitored with BPS or ferrozine.     

Organic Removal of Anaerobically Treated Leachate by Fenton Coagulation

The leachate from a Hong Kong landfill, containing 15,700 mg∕L of chemical oxygen demand (COD) and 2,260 mg∕L of ammonia nitrogen (NH3–N), was first treated in a UASB (upflow anaerobic sludge blanket) reactor at 37°C. The process on average removed 90.4% of COD with 6.6 days of hydraulic retention at an organic loading rate of 2.37 g of COD∕L?day. The UASB effluent was further treated by the Fenton coagulation process using H2O2 and Fe2+. Under the optimal condition of 200 mg of H2O2∕L and 300 mg of Fe2+∕L and an initial pH of 6.0, 70% of residual COD in the UASB effluent was removed, of which 56% was removed by coagulation∕precipitation and only 14% by free radical oxidation. It is obvious that H2O2 and Fe2+ had a strong synergistic effect on coagulation. The average COD in the final effluent was 447 mg∕L. Removing each gram of COD required 0.28 g of Fe2+ and 0.18 g of H2O2.     

Fenton-Like Oxidation of Polycyclic Aromatic Hydrocarbons in Soils Using Electrokinetics

An integrated electrochemical oxidation process that utilizes electrokinetics (EK) to deliver the oxidant (5–10% hydrogen peroxide, H2O2) and chelant [40 mM of ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA)] or iron chelate (1.4 mM Fe-EDTA or Fe-DTPA) to oxidize polycyclic aromatic hydrocarbons (PAHs) in soils was investigated. Batch and bench-scale EK experiments were conducted using: (a) kaolin, a low permeability clayey soil, spiked with phenanthrene at 500 mg/kg, and (b) former manufactured gas plant (MGP) soil, a high buffering silty soil, contaminated by a variety of PAHs (1493 mg/kg). Batch experiments showed that chelant solutions dissolve native iron minerals to form soluble Fe-chelates that remain available even at higher pH conditions of soil for the Fenton-like oxidation of the PAHs. In EK experiments, a 5–10% H2O2 solution was delivered from the anode and a chelant solution or iron-chelate was delivered from the cathode. Preflushing of soil with 5% ethanol and ferrous sulfate (1.4 mM) prior to oxidant delivery was also investigated. An electric potential of 2 VDC/cm was applied in all tests to induce electroosmotic flow for 5–8 days for kaolin and 25 days for the MGP field soil. In the absence of any chelating agent, phenanthrene oxidation was catalyzed by native iron present in kaolin soil, and 49.8–82.3% of phenanthrene was oxidized by increasing H2O2 concentration from 5–10%. At 5% H2O2 concentration, phenanthrene oxidation was not increased by using 40 mM EDTA, 40 mM DTPA or 1.4 mM Fe-DTPA, but it increased to 70% using 1.4 mM Fe-EDTA. Maximum phenanthrene oxidation (90.5%) was observed by 5% ethanol preflushing and then treating with 5% H2O2 at the anode and 1.4 mM Fe-EDTA at the cathode. However, preflushing with 1.4 mM ferrous sulfate did not improve phenanthrene oxidation. The results with the MGP field soil indicated that delivery of 5% H2O2 alone resulted in oxidation of 39.8% of total PAHs (especially 2- and 3-ring PAHs). The use of EDTA and Fe-EDTA did not increase PAHs oxidation in this soil. Overall, the results reveal that an optimized in situ combined technology of EK and Fenton-like process has the potential to oxidize PAHs in low permeability and/or high buffering soils.     

金属催化剂对Fenton试剂活性的影响因素分析

在分析Fenton氧化法作用机理的基础上,研究了金属催化剂Fe~(2+)/Fe~(3+)对Fenton试剂活性的影响。结果表明:Fe~(2+)和Fe~(3+)对Fenton试剂活性均具有催化作用,Fe~(2+)催化作用远大于Fe~(3+)催化作用;在H_2O_2投加量为12 mmol/L,反应温度为25℃,反应时间为25 min条件下,[Fe~(2+)]/[H_2O_2]的物质的量比为1∶10时,焦化含酚废水COD去除率最高达到90.2%(质量分数);在光照特别是紫外线协同作用下,Fe~(2+)和Fe~(3+)活性增大,且Fe~(3+)的活性增强趋势明显高于Fe~(2+)。     

Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis

In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.     

Na3AlF6-Al2O3熔盐中铝溶解度的测定

研究了铝在Na3AlF6-Al2O3熔盐中溶解度的测定方法。采用氟化钠与试样在高温下熔融,熔融物溶解水后加入硼酸-氢氧化钠溶液,过滤沉淀将氧化 铝除去,取其中一部分溶液在pH5.5～6.0 的条件下,加入过量的EDTA标准溶液,加热煮沸使之与铝完全络合,以二甲酚橙作指示剂,用锌标准溶液 滴定过量的EDTA ,从而得到Na3AlF6-Al2O3熔盐中铝离子的含量,再采用氟离子选择电极测定另一部分溶液中氟含量,计算得到冰晶石中铝离子含量 ,采用减氟法得到铝在Na3AlF6-Al2O33体系中溶解量。这个值是用来表征铝在电解质熔体中的溶解损失能力的大小,对生产工艺很重要。     

Kinetic analysis of the reaction between d(TTGGCCAA) and [Pt(NH3)3(H2O)]2+ by enzymatic degradation of the products and ESI and MALDI mass spectrometries

The kinetics of the reaction between the octanucleotide d(TTGGCCAA) in the single-stranded form in pure water and the platinum complex [Pt(NH3)3(H2O)]2+ was investigated by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometries coupled with enzymatic degradation of the adducts. These methods led to the determination of specific rate constants of platination. The global rate constant characteristic of the formation of adducts on each 5'- or 3'-guanine were measured by electrospray ionization analysis. The ratios between the 5'- and 3'-adducts were determined from enzymatic degradation of the final reaction mixture and MALDI analysis. The platination in water is approximately eight times faster than in 0.1 M NaClO4. The selectivity of platination is a factor of 2 in favor of the 5'-guanine, and similar to that observed for the reaction between d(CTGGCTCA) and [Pt(NH3)3(H2O)]2+ in 0.1 M NaClO4.