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A novel, highly sensitive superoxide dismutase biosensor for the direct and simultaneous determination of superoxide radicals was developed by immobilization of superoxide dismutase within carboxymethylcellulose-gelatin on a Pt electrode surface. The parameters affecting the performance of the biosensor were investigated. The response of the CMC-G-SOD biosensor was proportional to O (2) (·-) concentration and the detection limit was 1.25 × 10(-3) mM with a correlation coefficient of 0.9994. The developed biosensor exhibited high analytical performance with wider linear range, high sensitivity and low response time. The biosensor retained 89.8% of its sensitivity after use for 80 days. The support system enhanced the immobilization of superoxide dismutase and promoted the electron transfer of superoxide dismutase minimizing its fouling effect. The biosensor was quite effective not only in detecting O (2) (·-) , but also in determining the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues. 相似文献
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Ross A. Edwards Heather M. Baker M. M. Whittaker James W. Whittaker Geoffrey B. Jameson E. N. Baker 《Journal of biological inorganic chemistry》1998,3(2):161-171
The three-dimensional structure of the manganese-dependent superoxide dismutase (MnSOD) from Escherichia coli has been determined by X-ray crystallography at 2.1?Å resolution. The protein crystallizes with two homodimers in the asymmetric unit, and a model comprising 6528 protein atoms (residues 1–205 of all four monomers), four manganese ions and 415 water molecules has been refined to an R factor of 0.188 (R free 0.218). The structure shows a high degree of similarity with other MnSOD and FeSOD enzymes. The Mn centres are 5-coordinate, trigonal bipyramidal, with His26 and a solvent molecule, probably a hydroxide ion, as apical ligands, and His81, Asp167 and His171 as equatorial ligands. The coordinated solvent molecule is linked to a network of hydrogen bonds involving the non-coordinated carboxylate oxygen of Asp167 and a conserved glutamine residue, Gln146. The MnSOD dimer is notable for the way in which the two active sites are interconnected and a "bridge" comprising His171 of one monomer and Glu170 of the other offers a route for inter-site communication. Comparison of E. coli MnSOD and FeSOD (a) reveals some differences in the dimer interface, (b) yields no obvious explanation for their metal specificities, and (c) provides a structural basis for differences in DNA binding, where for MnSOD the groove formed by dimerization is complementary in charge and surface contour to B-DNA. 相似文献
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Jacques B. Baldensperger 《Archives of microbiology》1978,119(3):237-244
Superoxide dismutase has been purified to homogeneity from aerobically grown Thiobacillus denitrificans strain RT. It has a molecular weight of 43,000, is composed of two identical subunits which are not covalently bound, and contains 1.35 atom of iron per molecule. Absorption spectra and amino acid analysis are similar to those of other Fe-superoxide dismutases from bacteria. Aerobically and anaerobically grown cells contain the same Fe-enzyme with similar levels of activity. Manometric sulfite oxidation measurements suggest for the enzyme a protective function of sulfite against the autooxidation initiated by superoxide free radicals.Non-Standard Abbreviations DMSO
dimethyl sulfoxide
- SDS
sodium dodecyl sulfate
- SOD
superoxide dismutase 相似文献
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Superoxide dismutase was glycosidated with cyclodextrin-branched carboxymethylcellulose. The modified enzyme contained 1.4 mol polymer per mol protein and retained 87% of the initial activity. The anti-inflammatory activity of superoxide dismutase was 2.2-times increased after conjugation and its plasma half-life time was prolonged from 4.8 min to 7.2 h. 相似文献
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Membranes, which are an amalgam of proteins and lipids, effect electron transfer through largely unknown mechanisms. Using albumin with bound fatty acids as a model, we have investigated the possible role of these two membrane constituents in electron transfer. In the presence of albumin: fatty acid, there is substantial enhancement of the reduction of ferricytochrome C by ferrous iron. To assess the possible role of free superoxide in cytochrome C reduction, we added mammalian copper/zinc containing superoxide dismutase (Cu/Zn SOD), which catalyzes the transfer of electrons between superoxide anion radicals, forming oxygen and hydrogen peroxide. Surprisingly, in the presence of either albumin or fatty acid free albumin, Cu/Zn SOD actually accelerates electron transfer from ferrous iron to ferricytochrome C. By contrast, neither inactive Cu/Zn SOD nor active manganese SOD facilitates the ferrous iron-dependent reduction of cytochrome C. These results suggest that, in some circumstances, Cu/Zn SOD may transfer electrons to alternative acceptors and that such transfer depends upon the unique reduction/oxidation reaction mechanism of Cu/Zn SOD. If so, this ubiquitous enzyme could be involved in regulating cellular electron transfer reactions as well as acting as a superoxide 'detoxify-ing' agent. 相似文献
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A novel disposable biosensor based on direct electron transfer of superoxide dismutase (SOD) was fabricated for the determination
of superoxide anion. The biosensor was constructed by electrodeposition of gold nanoparticles (GNPs) on the indium tin oxide
(ITO) electrode and then immobilization of SOD in silica sol–gel (SG) network in the presence of cysteine on GNPs/ITO modified
electrode surface. The distribution of GNPs on ITO electrode surface was examined by scanning electron microscopy (SEM). The
immobilized SOD exhibited high catalytical activity towards superoxide anion. Parameters affecting the performance of the
biosensor were also investigated. A linear calibration curve was obtained over the range from 0.08 to 0.64 μM with a correlation
coefficient of 0.9937. The resulted biosensors were demonstrated to possess striking analytical properties for superoxide
anion determination, such as high sensitivity, good accuracy, and long-term stability. It provides a promising platform for
the fabrication of disposable biosensors. 相似文献
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More than 100 different mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS)--a fatal neurodegenerative disease in which aggregation of the SOD1 protein is considered to be the primary mode of pathogenesis. Recent results show that these mutations have remarkably diverse and unexpected effects on the structure, activity and native state stability of SOD1. Intriguingly, many mutations seem to have no measurable effect on the biophysical and biochemical properties of SOD1, except for decreasing the net charge of the protein. Thus, it seems likely that different ALS-associated mutations promote SOD1 aggregation by fundamentally distinct mechanisms. Understanding this complexity has implications for drug development and treatment of the disease. 相似文献
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Summary The proposed transfer of the gene for Cu/Zn superoxide dismutase from the ponyfish to its symbiotic bacteriumPhotobacterium leiognathi has been evaluated by an extensive analysis of all available Cu/Zn superoxide dismutase sequences. By the use of four different computer programs, phylogenetic trees were constructed from the sequences of the superoxide dismutases of human, ox, pig, horse, swordfish, fruit fly, yeast, andNeurospora crassa to find out whether superoxide dismutase sequences can reliably be used for the reconstruction of genealogical relationships. All programs arrived at the same most parsimonious tree (one requiring 232 amino acid replacements), the topology of which conformed to established opinions about the phylogenetic relations among these eukaryotes, except that it placed humans closer to the artiodactyls ox and pig than it placed horses. This could be corrected at the cost of two amino acid replacements. The sequence ofP. leiognathi superoxide dismutase was then connected at all possible positions to the corrected eukaryotic tree. It was slighly more parsimonious to link the bacterial sequence to the root of the tree than to the fish branch: The former required 316 (or 317) amino acid replacements, versus 319 for the latter. This relative lack of discrimination between such distinct alternative topologies may be a general complication in the comparison of prokaryotic and eukaryotic proteins: Bacterial cytochrome c sequences also were found to be connected as parsimoniously to the root of the eukaryotic tree as to any terminal or ancestral branch. It was calculated that the rate of evolution of the bacterial superoxide dismutase gene, if transfer occurred 30 million years (Myr) ago, must have amounted to 487 amino acid replacements per 100 residues per 100 Myr. This is more than 5 times the highest rate observed in any protein (that found for fibrinopeptides), and even much higher than the maximum rate of protein evolution that can be deduced from the neutral mutation rate of unconstrained DNA. Also, no significant evidence that shared derived amino acid replacements are present in swordfish andP. leiognathi superoxide dismutase, as might be expected had gene transfer occurred, was found. On the basis of the available data it seems more reasonable to ascribe the isolated occurrence of Cu/Zn superoxide dismutase inP. leiognathi (as well as inCaulobacter crescentus) to irregular patterns of gene expression and inactivation in the course of divergent evolution than to undocumented processes of gene transfer from eukaryotes to prokaryotes. 相似文献
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Kevin W. Sea Yuewei Sheng Herman L. Lelie Lindsay Kane Barnese Armando Durazo Joan Selverstone Valentine Edith Butler Gralla 《Journal of biological inorganic chemistry》2013,18(8):985-992
Copper–zinc superoxide dismutase (Sod1) is an abundant intracellular enzyme that catalyzes the disproportionation of superoxide to give hydrogen peroxide and dioxygen. In most organisms, Sod1 acquires copper by a combination of two pathways, one dependent on the copper chaperone for Sod1 (CCS), and the other independent of CCS. Examples have been reported of two exceptions: Saccharomyces cerevisiae, in which Sod1 appeared to be fully dependent on CCS, and Caenorhabditis elegans, in which Sod1 was completely independent of CCS. Here, however, using overexpressed Sod1, we show there is also a significant amount of CCS-independent activation of S. cerevisiae Sod1, even in low-copper medium. In addition, we show CCS-independent oxidation of the disulfide bond in S. cerevisiae Sod1. There appears to be a continuum between CCS-dependent and CCS-independent activation of Sod1, with yeast falling near but not at the CCS-dependent end. 相似文献
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《Gene》1996,179(1):33-37
Xanthomonas showed atypical regulation of catalase (Kat) and superoxide dismutase with respect to growth phase and response to various inducers. The highest levels of both enzymes were detected during early log phase of growth and declined as growth continued. This was in contrast to resistance levels to superoxides, H2O2 and organic peroxides, which reached maximum levels during stationary phase. Xanthomonas catalase was induced over six fold by superoxide generators and methyl methane sulfonate but weakly by H2O2. The regulation pattern of these enzymes could be important during plant/microbe interactions. To facilitate elucidation of Xanthomonas kat gene regulation, highly conserved regions of monofuctional Kat amino acid sequences were used to synthesize oligodeoxyribonucleotide primers for use in PCR reactions with Xanthomonas genomic DNA as templates. The Xanthomonas-specific PCR kat probe was used to isolate a functional kat from Xanthomonas campestris pv. phaseoli. 相似文献
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M. A. Z. Ayub S. Astolfi-Filho F. Mavituna S. G. Oliver 《Applied microbiology and biotechnology》1992,37(5):615-620
Summary A double mutant sod1/pgk1 strain of Saccharomyces cerevisiae has been constructed in order to investigate the effects of different environmental conditions on yeast physiology, plasmid stability, and superoxide dismutase (SOD) production. Strains were transformed with yeast episomal plasmids (YEp) containing both PGK1 and SOD1 genes and were grown on fermentable carbon sources and under vigorous aeration. Under these conditions, the presence of the PGK1 gene was made essential for growth and both genes were efficiently expressed. However, plasmid-borne PGK1 was found not to increase the stability of YEp vectors in batch cultures of Pgk– cells. Paradoxically, plasmid stability increased during the respiratory phase of growth. An investigation of the metabolism of Pgk– cells demonstrated that these glycolytic pathway mutants do not appreciably metabolize glycerol. Thus Pgk+, plasmid-containg, cells have a selective advantage during the respiratory phase of batch growth since they can utilize both glycerol and ethanol.
Correspondence to: S. G. Oliver 相似文献
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Marta Ferraroni Wojciech R. Rypniewski Bruno Bruni Pierluigi Orioli S. Mangani 《Journal of biological inorganic chemistry》1998,3(4):411-422
The crystal structures of dithionite-reduced bovine Cu(I),Zn superoxide dismutase and of its adducts with the inorganic anions azide and thyocyanide have been determined in a C2221 crystal form obtained at pH?5.0. This crystal form is characterized by a high solvent content (72%) and by having the two Cu,ZnSOD monomers (A and B) in different crystal environments. One of them (B) is involved in few intermolecular crystal contacts so that it is in a more "solution like" environment, as indicated by average temperature factors which are about twice those of the other monomer. The differences in crystal packing affect the active site structures. While in the A monomer the Cu(I) is coordinated to all four histidine residues, in the B monomer the bridging His61 side chain is found disordered, implying partial detachment from copper. The same effect occurs in the structures of the anion complexes. The inorganic anions are found bound in the active site cavity, weakly interacting with copper at distances ranging from 2.5 to 2.8?Å. The copper site in the A subunit of the native enzyme structure displays significant electron density resembling a diatomic molecule, bound side-on at about 2.8?Å from the metal, which cannot be unambiguously interpreted. The crystallographic data suggest that the existence of the His61 bridge between copper and zinc is dominated by steric more than electronic factors and that the solution state favors the His61 detachment. These structures confirm the existence of an energetically available state for Cu(I) in Cu,ZnSOD where the histidinato bridge to zinc is maintained. This state appears to be favored by tighter crystal contacts. The binding of the anions in the active site cavity is different from that observed in the oxidized enzyme and it appears to be dominated by electrostatic interactions within the cavity. The anion binding mode observed may model the substrate interaction with the reduced enzyme during catalysis. 相似文献
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Danielsson J Kurnik M Lang L Oliveberg M 《The Journal of biological chemistry》2011,286(38):33070-33083
Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble. 相似文献
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《Biochimica et Biophysica Acta (BBA)/General Subjects》2002,1569(1-3):151-158
The direct electrochemical redox reaction of bovine erythrocyte copper–zinc superoxide dismutase (Cu2Zn2SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65±3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant ks is 1.2±0.2 s−1 and the anodic (αa) and cathodic (αc) transfer coefficients are 0.39±0.02 and 0.61±0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu2Zn2SOD), its Cu- or Zn-free derivatives (E2Zn2SOD and Cu2E2SOD, E designates an empty site) and the SOD reconstituted from E2Zn2SOD and Cu2+. The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion. 相似文献
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Superoxide dismutase can either inhibit or stimulate autoxidation of different hydroquinones, suggesting multiple roles for O2.-. Inhibitory actions of superoxide dismutase include termination of O2.(-)-propagated reaction chains and metal chelation by the apoprotein. Together, chelation of metals and termination of O2.(-)-propagated chains can effectively prevent reduction of oxygen. Chain termination by superoxide dismutase can thus account for negligible accumulation of H2O2 without invoking a superoxide:semiquinone oxidoreductase activity for this enzyme. One stimulatory action of superoxide dismutase is to decrease thermodynamic limitations to reduction of oxygen. Whether superoxide dismutase inhibits or accelerates an autoxidation depends on the reduction potentials of the quinone and the availability of metal coordination for inner sphere electron transfers. 相似文献