Microcalorimetric studies on the dismutation of superoxide anion catalyzed by superoxide dismutase

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Determination of superoxide dismutase activity with an electrochemical oxygen probe

A flow system is described for measurements of superoxide dismutase activities over wide concentration ranges by varying the substrate, pH and flow conditions. Pyrogallol and 6-hydroxydopamine were the best substrates found; the limits of detection were 1.5×10^?9 M superoxide dismutase at pH 9.5 and 2×10^?10 M at pH 7.4, respectively. Epinephrine was less suitable; catechol was not useful. Epinephrine provided good sensitivity at pH 10 when a residence time of 8 min in the system was allowed, but the measurements were then less reproducible than with pyrogallol or 6-hydroxydopamine.     

Determination of superoxide dismutase in erythrocytes by a chemiluminescent assay

A highly rapid chemiluminescent assay for the determination of superoxide dismutase (SOD) activity in erythrocytes was developed. The inhibition of the luminescent emission caused by the decrease of generated superoxide anions was measured. The aim of this work was to verify the application of a non amplified luminol SOD luminescent assay (CLM) in erythrocytes starting from an amplified method already used for the determination of XOD activity in milk (CLME). Both the assays had a detection limit of 3x10(-2)+/-7x10(-3) U/ml of SOD at 2sigma level, and a linear range of activity from 5.2 to 0.03 U/ml of SOD. The imprecision of assays (repeatability) presented coefficients of variations ranging from 3.1 to 7.9% for the CLME method and from 0.6 to 17.7% for CLM method. Both luminescent techniques were compared using a spectrophotometric kit, that had a detection limit of 0.3 U/ml, and showed good agreement.     

Theoretical mechanisms of the superoxide radical anion catalyzed by the copper‐zinc superoxide dismutase

The disproportionation of superoxide radical anions catalyzed by copper‐zinc superoxide dismutase was investigated in detail using density functional theory. The structures of each stationary point and the transition states were located so that the reaction pathways were determined. The results indicate that the reactions proceed by two steps both for the oxidized process of superoxide radical anion and the reduced one. The rate for the determining step of the reaction (2) is in very good agreement with the experimental value. The solvation effect on the reaction was also discussed. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

Mechanisms of electron transfer in catalysis by copper zinc superoxide dismutase

Activated oxygen intermediates during copper zinc superoxide dismutase (SOD) catalysis were investigated using an isotope fractionation technique and natural abundance reagents. Competitive oxygen kinetic isotope effects (KIEs) are reported for the enzyme-catalyzed disproportionation of superoxide as well as the stoichiometric reaction of reduced SOD with molecular oxygen. Analysis within the context of quantum mechanical electron transfer theory provides evidence against an outer-sphere mechanism for O2*- oxidation. A CuII-O2-I intermediate is, therefore, proposed. The SOD-catalyzed oxidation of O2*- is characterized by an inverse (<1) KIE which is similar to those determined for the analogous reactions of synthetic copper compounds. An inverse kinetic isotope effect upon the enzymatic reduction of O2*- is also observed and proposed to arise from rate-determining proton transfer which leads to the formation of HO2* in the SOD active site.     

Molecular dynamics simulations of the enzyme Cu, Zn superoxide dismutase

The enzyme Cu, Zn superoxide dismutase (Cu,Zn-SOD) is a ubiquitous oxireductase, which is responsible for the cellular defense against oxidative stress caused by the high toxicity of the superoxide radical, and has been also linked to some cases of familiar amyotrophic lateral sclerosis. In the present study a set of molecular mechanics parameters for the active site of Cu,Zn-SOD has been derived. Afterward, an extensive molecular dynamics simulation has been carried out in an aqueous environment. The obtained results shed a further light on the structural flexibility of the backbone, where the active site is nested, and the solvation shell occupancy. The relatively small backbone deviation, shown by a root-mean-square deviation below 1.0 A, confirms the accuracy of the parameters. The solvent shell analysis has shown that the first solvation shell is located at about 5 A from the copper ion, generating an empty cavity with enough space to accommodate the superoxide radical. The low residence time means that a high permutation rate of water molecules in both solvation shells is consistent with the efficiency of this catalytic mechanism. Hybrid studies using ONIOM methodologies can now be done to evaluate the mechanistic implications of the explicit inclusion of the whole system.     

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.     

超氧化物歧化酶模型化合物的合成, 表征和活性测定

合成了二(2-苯并咪唑亚甲基)胺(N3)及其四种全新的过渡金属的双核配合物。通过元素分析、红外光谱和紫外光谱对配体及配合物进行了结构表征,利用邻苯三酚自氧化法测定了四种模拟化合物催化超氧阴离子自由基歧化反应的活性。     

Photohemolysis of erythrocytes enriched with superoxide dismutase, catalase and glutathione peroxidase

Abstract— Cationic liposomes have been used to introduce into human erythrocytes variable amounts of the enzymes superoxide dismutase, catalase and glutathione peroxidase. The behaviour of the enzyme-enriched erythrocytes toward photohemolysis sensitized by protoporphyrin IX has been studied in comparison to that of erythrocytes treated with empty liposomes. The erythrocytes with increased catalase and glutathione peroxidase activity were more resistant to photohemolysis. In contrast, the erythrocytes enriched with superoxide dismutase hemolyzed faster. The association of two enzymes at a time was also studied     

Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography.

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu(2+)-IMAC column with an increasing gradient of a counter ion (NH+4) run in combination with an increasing pH gradient (6.8-7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.     

绿豆萌发过程中SOD的分离纯化及性质研究

制备不同萌发时期绿豆SOD(超氧化物歧化酶)粗酶液,筛选出酶比活力最高的时期,并运用热变性、硫酸铵分级、DEAE-Sepharose FF阴离子交换层析和Sephacryl S-100 HR分子筛层析分离纯化并作对比考察,提纯的SOD进行了理化性质研究。结果表明,绿豆种子萌发第3天时SOD的比活力最高,经纯化获得的SOD酶比活力为2256.4 U/mg,纯化倍数为3.9。绿豆中SOD为Cu/Zn SOD和Mn SOD,分子量为68KD,热稳定性高,在pH5-8条件下稳定,对高浓度SDS具有抗性。EDTA可以一定程度上抑制SOD活性,H2O2可以完全抑制SOD活性。     

Determination of metal content in superoxide dismutase enzymes by capillary electrophoresis†

Superoxide dismutases are antioxidant scavenger enzymes that contain a metal cofactor (copper, zinc, iron, and manganese) in their active site. Metal content measurement is one of the essential steps to characterize enzyme biological activity. We have developed a capillary electrophoretic protocol for the determination of the metal content in superoxide dismutase enzymes. The background electrolyte containing 10 mM pyridine‐2,6‐dicarboxylic acid and 1 mM 1‐methyl‐3‐tetradecylimidazolium chloride at pH 3.8 was optimized for on‐column complexation of the above‐mentioned metals. The minimum detectable levels of metals ranged from 0.3 to 1.2 μg/mL. The reliability of the method was checked by parallel quantitative determination of the metal content in superoxide dismutase enzymes by graphite furnace or flame atomic absorption spectrophotometry methods.     

Kinetic study of nickel-thiolate oxygenation by hydrogen peroxide. Implications for nickel-containing superoxide dismutase

The rate of metal-thiolate oxygenation by hydrogen peroxide has been measured for a model complex of nickel-containing superoxide dismutase (NiSOD) as 0.47 ± 0.03 M(-1) s(-1) at 303 K. Under typical synthetic conditions the reaction proceeds within seconds yielding S-oxygenate products. At biologically relevant H(2)O(2) concentrations, half-life times extend to several hours or days. The results suggest H(2)O(2) oxidation of NiSOD is not kinetically competent on the SOD timescale accounting for the lack of S-oxygenation byproducts.     

Photoinduced hydrogen evolution in an artificial system containing photosystem I,hydrogenase,methyl viologen and mercaptoacetic acid

Hydrogen evolution was detected in an artificial system composed of light-harvesting unit of purified photosystem Ⅰ, catalyst ofhydrogenase, methyl viologen and electron donor under radiation. Absorption spectral features confirmed that electron transferfrom electron donors to proton was via a photoinduced reductive process of methyl viologen.2008 Dong Jin Qian. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.