GENERATION AND PARTICIPATION OF O-2 IN 2-NITROPROPANE DIOXYGENASE REACTION

Abstract— 2-Nitropropane dioxygenase (EC 1. 13. 11) of the yeast Hansenula mrakii catalyzes the oxygenative denitrification of 2-nitropropane as follows:

The enzyme is significantly inhibited by superoxide dismutase and various scavengers for superoxide such as cytochrome c , epinephrine, thiols and polyhydric phenols. The scavengers added to the reaction mixture were oxidized or reduced. The addition of superoxide dismutase and the omission of 2-nitropropane or oxygen prevented the oxidation and the reduction of the scavengers. The enzyme catalyzes the formation of nitrite from 2-nitropropane by KO₂ added anaerobically.
One mole of NADH is bound per mole of the enzyme and predominantly the pro-R hydrogen of bound NADH is transferred to superoxide formed enzymatically or provided externally. The enzyme shows incomplete stereospecificity for hydrogen transfer from NADH.     

Zinc deficient bovine erythrocyte superoxide dismutase has low specific activity.

Zinc deficient bovine superoxide dismutase (Cu2E2SOD (E = empty)) was prepared and purified by high performance liquid chromatography (HPLC). Each peak was characterized as to protein, copper content and specific activity. The Cu2E2SOD peak fractionated by HPLC has a low specific activity at pH 7.8 (about 10% of the native enzyme (Cu2Zn2SOD)). With the addition of zinc ions, the specific activity of Cu2E2SOD was quantitatively restored to that of the native enzyme. This behavior implies that the zinc ion is very important for the appearance of enzyme activity.     

Development of a new biosensor for superoxide radicals

A superoxide dismutase (SOD) biosensor for determination of superoxide radicals has been developed by immobilization of superoxide dismutase within gelatin (G) on a Pt electrode surface. The properties of the biosensor have been investigated and optimum conditions–enzyme concentration, glutaraldehyde concentration, and pH–were determined. The response of the G-SOD biosensor was proportional to concentration and the detection limit was 0.01 mmol L⁻¹ at a signal-to-noise ratio of 3. The biosensor retained 89% and 60% of its sensitivity after use for three and four weeks, respectively. Immobilization of SOD on gelatin provides a biocompatible microenvironment around the enzyme and stabilizes the activity of the enzyme very efficiently. The superoxide dismutase biosensor was used to determine the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues.     

PROTECTIVE EFFECT OF SELENIUM AND ZINC ON UV-A DAMAGE IN HUMAN SKIN FIBROBLASTS

Ultraviolet A radiation participates in cytotoxicity and carcinogenesis of the skin by a mechanism involving the generation of reactive oxygen species. Endogenous antiradical defense systems utilize metalloenzymes including Se-dependent glutathione peroxidase and Cu and Zn superoxide dismutase. The aim of the present work was to determine the protective effect of two trace elements, Se and Zn, on cultured human diploid fibroblasts exposed to UV-A radiation (broad-spectrum source with a maximum intensity at 375 nm). Selenium in the culture medium (0.1 mg/L) in the form of sodium selenite increased the synthesis and activity of glutathione peroxidase by 60.5% in the absence of exposure to UV-A radiation and by 35% after irradiation with 5 J/cm² ( P = 0.043). The presence of this element significantly increased the survival of UV-A-irradiated fibroblasts ( P < 0.0001). This confirms the essential role of Se in the detoxifying activity of the enzyme. In addition, thiobarbituric acid-reacting substances (TBAR), which are lipid peroxidation markers, decreased in the presence of exogenous Se:—19% and -22% without irradiation and after irradiation with 5 J/cm² ( P = 0.056). When Zn was added at the dose of 6.5 mg/L as ZnCl₂, fibroblasts subjected to oxidizing stress induced by UV-A were protected from cytotoxicity ( P <0.0001). The TBAR production decreased significantly: -33% without irradiation and -34% after irradiation with 5 J/cm² ( P = 0.008). Superoxide dismutase activity, however, decreased after supplementing with Zn: - 26% without irradiation and - 20% after UV-A irradiation ( P = 0.017). The antioxidant properties of Zn are thus apparently independent of superoxide dismutase activity.     

Enzymatic activity enhancement of non-covalent modified superoxide dismutase and molecular docking analysis

The enzyme activity of superoxide dismutase was improved in the pyrogallol autoxidation system by about 27%, after interaction between hydroxypropyl-β-cyclo-dextrin and superoxide dismutase. Fluorescence spectrometry was used to study the interaction between hydroxypropyl-β-cyclodextrin and superoxide dismutase at different temperatures. By doing this, it can be found that these interactions increase fluorescence sensitivity. In the meantime, the synchronous fluorescence intensity revealed the interaction sites to be close to the tryptophan (Trp) and tyrosine (Tyr) residues of superoxide dismutase. Furthermore, molecular docking was applied to explore the binding mode between the ligands and the receptor. This suggested that HP-β-CD interacted with the B ring, G ring and the O ring and revealed that the lysine (Lys) residues enter the nanocavity. It was concluded that the HP-β-CD caused specific conformational changes in SOD by non-covalent modification.     

Superoxide dismutase activity in some strains of lactobacilli: induction by manganese

Dialyzed cell-free extract of lactobacilli was found to contain superoxide dismutase activity by using a test system in which superoxide ion is generated by xanthine oxidase. The specific activities of Lactobacillus acidophilus ATCC 4356, Lactobacillus murinus ATCC 35020, Lactobacillus acidophilus CRL 358, Lactobacillus plantarum ATCC 8014, Lactobacillus casei CRL 431, Lactobacillus plantarum CRL 353, Lactobacillus fermentum ATCC 9338, Lactobacillus buchneri NCDO 110, and Lactobacillus fermentum CRL 251 were between 0.06 and 0.43 U/mg protein. The presence of superoxide dismutase activity was demonstrated when the strains were grown in media containing Mn2+ ions. Superoxide dismutase of lactobacilli may be an Mn enzyme since it was not inhibited by either cyanide or azide ions. However, the cell-free extract of Lactobacillus murinus ATCC 35020 contains superoxide dismutase activity sensitive to both ions.     

SOD mimetic activity of salicylatocopper complexes

Investigation of salicylatocopper complexes in the presence of a nitrogen donor ligand is a growing research area due to the interesting mimetic activities of such complexes. Here, three X-salicylatocopper (where X = 3-methyl, or 4-methoxy) complexes with three different N-donor ligands, [Cu(μ-menia)(3-Mesal)₂(menia)(H₂O)]₂ (I), Cu(3-Mesal)₂(denia)₂(H₂O)₂ (II), Cu(4-MeOsal)₂(2-pyme)₂ (III), are presented (where 3-Mesal = 3-methysalicylate, 4-MeOsal = 4-methoxysalicylate, menia = N-methylnicotinamide, 2-pyme = 2-pyridylmethanol). The complexes were characterized by elemental analysis, IR and UV-VIS spectrophotometry. Cyclic voltammetry and the superoxide dismutase activity of the prepared complexes in solution were measured and the complexes were characterized by means of the inhibition concentration IC₅₀. In addition, the superoxide dismutase (SOD) activity of these complexes was compared with those of the parent ligand copper acetate, native SOD enzyme, and the related copper complexes containing non-steroidal anti-inflammatory drugs. The resulting SOD activity was correlated to the composition, structure and redox stability of the prepared complexes. The best value of the inhibition concentration was found for complex I (IC₅₀ = 2.24 µM), which classifies this complex into a group of good superoxide scavengers.     

利用高效弱阴离子交换色谱法纯化超氧化物歧化酶

利用高软弱阴离子交换色谱法简化了超氧化物歧化酶（ＳＯＤ）的纯化手续。在丙酮沉淀后,仅用一步色谱分离就能使来自牛血的Ｃｕ,Ｚｎ－ＳＯＤ达到电泳纯,活性回收率为８６．４％,比活为７７１１Ｕ／ｍｇ,纯化倍数提高至５２倍。此外,详尽讨论了色谱分离的条件。     

固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶

以Ｃｕ~（２＋）－Ｓｅｐｈａｒｏｓｅ４Ｂ固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶,考察了不同的洗脱缓冲溶液及其ｐＨ对纯化效率的影响,显示了方法的有效性。     

Chemical composition and antioxidant activity of sulphated polysaccharides extracted from Fucus vesiculosus using different hydrothermal processes

Sulphated polysaccharides (SP) were extracted from Fucus vesiculosus seaweed by using two different hydrothermal processes: microwave-assisted extraction (MAE) and autohydrolysis (AH). The extraction yields, chemical composition, and antioxidant activity of the polysaccharides extracted were determined and compared. Although both processes afforded SP with similar yields (18.2 mass % and 16.5 mass %, for MAE and AH, respectively) and l-fucose as the main monosaccharide, the heterogeneous structure of the polysaccharide recovered was significantly affected by the AH process. The SP obtained by MAE contained 53.8 mole % of fucose, 35.3 mole % of xylose, and 10.8 mole % of galactose; while the SP obtained by AH was composed of 76.8 mole % of fucose and 23.2 mole % of galactose. Both samples presented comparable values of antioxidant activity by the di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (2,2-diphenyl-1-picrylhydrazyl, DPPH), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and lipid oxidation inhibition methods, but the polysaccharide obtained by AH exhibited a higher antioxidant potential by the differential pulse voltammetry technique. This study demonstrates that the chemical composition and antioxidant activity of SP obtained from F. vesiculosus vary according to the process used for their extraction. However, the SP obtained by MAE or AH both have the potential for use as natural antioxidants in industrial applications.     

PEDOT/Superoxide Dismutase Electrode Surface Modification for Superoxide Bioelectrochemical Sensing

An amperometric biosensor for the sensitive detection of superoxide was designed utilizing a drop‐coating approach for immobilizing the superoxide dismutase enzyme on Pt electrode modified with a thin layer of poly (3,4‐ethylenedioxythiophene) (PEDOT). The layer electrodeposited on Pt was characterized by cyclic voltammetry and atomic force microscopy (AFM). Then, drop‐coating procedure was chosen for the immobilization of superoxide dismutase (SOD), which was incorporated at the electrode surface using a solution containing SOD, glutaraldehyde and bovine serum albumin (optimized composition: SOD 0.1 % – BSA 2 % – GA 2.5 %.) This simple procedure allows forming a reproducible enzymatic biocomposite layer that allows optimal sensitivity and limit of detection for superoxide sensing. The synergistic effect integrates an effective conductivity and permselectivity attributed to the PEDOT layer, as well as the specificity and selectivity of SOD for the detection of superoxide. A high sensitivity (0.82±0.01 μA/μM) and a low detection limit of 11 nM were obtained, as well as good selectivity against main interfering biological compounds such as uric acid and ascorbic acid. Our results suggest that the biosensor could be used for the detection and quantification of in vitro and in vivo.     

Purification of pro- and eukaryotic superoxide dismutases by charge-controlled hydrophobic chromatography

The process of purifying superoxide dismutases was simplified using charge-controlled hydrophobic chromatography on 10-carboxydecyl Sepharose. In only one chromatographic step following ammonium sulphate precipitation, Fe-containing superoxide dismutase from Pseudomonas putida and Cu,Zn-containing superoxide dismutase from bovine erythrocytes were purified with an overall yield of about 70% to electrophoretic homogeneity. The specific activities of the crystalline enzyme preparations were expressed in McCord and Fridovich units and were 3000 and 3200 U/mg, respectively.     

Study on the interaction of copper-zinc superoxide dismutase with aluminum ions by electrochemical and fluorescent method

The interaction of superoxide dismutase (SOD) with aluminum (Al) ions was investigated by cyclic voltammetry, fluorescence spectroscopy and synchronous fluorescence spectroscopy. The electrochemical activity of the SOD enzyme electrode was inhibited irreversibly by the addition of Al. Meanwhile, the static fluorescence quenching mechanism further revealed the existing of molecular complex of SOD with Al(3+). The association constant was obtained from Lineweaver-Burk plot. The experimental results of voltammetry and fluorescence spectroscopy indicated that the conformation of SOD molecule was altered by the formation of Al-SOD complex. It may influence the activity of SOD enzyme since the optimum action of SOD depends upon a particular configuration of electrostatic charges in the enzyme molecule.     

Detection of synergistic effect of superoxide dismutase and jujubosides on scavenging superoxide anion radical by capillary electrophoresis

A new capillary electrophoresis method was developed to study the synergistic effect of superoxide dismutase and jujuboside A or B on scavenging superoxide anion radical in serum matrix respectively, in which superoxide anion radical was generated from pyrogallol autoxidation. The electrophoresis conditions, and the factors affecting the productive rate of purpurogallin, such as pyrogallol autoxidation product and the activity of superoxide dismutase, were optimized. Under optimal conditions, the content of superoxide dismutase in Gibco newborn calf serum was 7.06 mg/L, RSD was 2.01% and the average recovery was 98.4%. The values of IC₅₀ for jujuboside A and B in the serum matrix were 157.67 and 31.60 mg/L respectively, and they both had synergy on scavenging superoxide anion radical with superoxide dismutase, but there was no the dose‐dependency on this synergy.     

人CuZn-SOD的分子改造及在毕赤酵母中的表达

为了改善人CuZn-SOD的酶学性质, 在借鉴人细胞外超氧化物歧化酶的特性基础上, 利用分子生物学技术, 在人CuZn-SOD的C末端加入肝素亲和肽构建了人CuZn-SOD工程酶, 并在毕赤酵母中获得表达, 经纯化的工程酶具有较强的肝素亲和性.     

Crystal structure of the first SH-containing tetrahedral cobalt(II) complex, [Co(quinoline)2(SH)2]. Superoxide dismutase activity

The preparation, spectroscopic properties and crystal structure of the bis-quinoline–bis-mercaptocobalt(II) complex [Co(quinoline)₂(SH)₂] are reported. The complex is tetrahedral with nitrogen donor atoms from two quinoline ligands and sulphur donor atoms from two mercapto groups. The superoxide dismutase mimetic activity of the complex was investigated using the indirect xanthine–xanthine oxidase–nitroblue tetrazolium method and compared to that of the native enzyme.     

β-CD-Shiff碱铜(Ⅱ)配合物模拟酶催化光度法的研究

合成了Shiff碱2-羟基-1-萘醛缩-2-氨基噻唑(HNATS)及其铜配合物,发现配合物Cu(Ⅱ)-(HNATS)~2具有显著的过氧化物模拟酶活性,可催化H~2O~2氧化4-氨基安替比林与2,4-二氯苯酚偶联反应。研究了β-环糊精对模拟酶催化活性的影响,探讨了反应机理,考察了反应条件和共存物质的影响,建立了新的测定超氧阴离子自由基(O~2^-^.)的分光光度法,线性范围为0～5.0×10^-^4mol/L,检出限为5.0×10^-^6mol/L。方法用于测定人体血液中超氧化物歧化酶(SOD)活性,结果满意。     

CHANGES IN SUPEROXIDE DISMUTASE, CATALASE AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITIES OF RABBIT ALVEOLAR MACROPHAGES: INDUCED BY POSTNATAL MATURATION AND/OR IN VITRO HYPEROXIA

Abstract— The total superoxide dismutase activity of rabbit alveolar macrophages (AM) increased twofold during the first postnatal week. This increase in superoxide dismutase activity was primarily mitochondrial and paralleled the increase in the number of mitochondria in these cells which has been previously reported. The superoxide dismutase activity of AM in culture for 18 h was significantly increased by hyperoxia; catalase activity in hyperoxic conditions was slightly adversely affected; glucose-6-phosphate dehydrogenase activity was increased but not significantly over control values. Hyperoxic cultures beyond 42 h decreased the total number of viable cells and the superoxide dismutase activity expressed per viable cell; the disappearance of catalase and glucose-6-phosphate dehydrogenase activities in this period, however, was more rapid in control alveolar macrophages than those under hyperoxia.     

Xanthine oxidase-assisted catalysis by dopamine β-hydroxylase: Mechanistic considerations on the role of superoxide anion

Dopamine β-monooxygenase catalyzes the transformation of dopamine into norepinephrine by inserting an O-atom on a benzylic C–H bond. The activation of O₂ occurs at a copper-containing active site in the presence of a reducer (ascorbate) that enables that copper ions be reduced to Cu(I) and reoxidized during catalysis. In the present paper, we establish that the xanthine/xanthine oxidase coupled system is a cofactor for this enzyme, and that hydroxylation of substrate tyramine is time-dependent. Using superoxide dismutase, we unambiguously prove that the species responsible for the hydroxylase activity is superoxide anion. The optimum pH for this activity is 6.8, a value about one pH unit higher than the physiological pH for the enzyme. Moreover, we propose a mechanism that takes into account all of our results, and describes putative interactions between the copper ions of the active site and superoxide anion.     

Crystal structure and some properties of a novel potent Cu2Zn2SOD model schiff base copper(II) complex ∗[Cu(bppn)](ClO4)2∗2 · H2O

The compound *[Cu(bppn)](ClO₄)₂*₂ · H₂O (where BPPN = N,N′-propylenebis[2-benzoylpyridineiminato]) was prepared by reaction of 2-benzoylpyridine and 1,3-propanediamine with copper perchorate in ethanol and characterised by X-ray crystallography. The central Cu atom in two crystallographical non-equivalent [Cu(bppn)]²⁺ cations exhibited a slightly distorted tetrahedral geometry with the Cu---N bond distances of 1.967(5)–2.010(5) Å and 1.966(5)–2.000(5) Å, respectively. The electronic absorption ₆₄₆ was 194 M⁻¹ cm⁻¹. The EPR parameters g = 2.051 and g = 2.226 were in accordance with the respective values of intact Cu₂Zn₂ superoxide dismutase (Cu₂Zn₂SOD). Its electronic property displayed a single quasiversible one—electron reduction process at − 0.204 V with ΔE_p = 84 mV, suggesting the title complex possesses high superoxide dismutase (SOD) activity.