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.     

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     

Bienzymatic supramolecular complex of catalase modified with cyclodextrin-branched carboxymethylcellulose and superoxide dismutase: stability and anti-inflammatory properties

A bienzymatic supramolecular assembly of CAT and SOD is reported. CAT was chemically glycosilated with CD branched CMC and then associated with SOD modified with 1-adamantane carboxylic acid. SOD was remarkably resistant to inactivation by H(2)O(2) and its anti-inflammatory activity was 4.5-fold increased after supramolecular association with the modified CAT form. [structure: see text]     

Liposome-recruited activity of oxidized and fragmented superoxide dismutase

The peptide fragment of H2O2-treated Cu,Zn-superoxide dismutase (SOD) was found to be reactivated with liposomes prepared by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The fragmentation of SOD was observed by 2 mM H2O2 treatment as well as by SOD inactivation and the loss of an alpha-helix in the neighborhood of its activity center. The H2O2-treated SOD, which lost its activity at different incubation times, was dramatically reactivated only by adding POPC liposomes, resulting in 1.3-2.8 times higher enzymatic activity. The ultrafiltration analysis of H2O2-treated SOD co-incubated with liposomes shows that some specific peptide fragments of the oxidized SOD can interact with POPC liposomes. A comparison of the fractions detected in reverse-phase chromatography shows that specific SOD fragments are able to contribute to the reactivation of oxidized and fragmented SOD in the presence of POPC liposomes. The liposomes can recruit the potentially active fragment of SOD among the lethally damaged SOD fragments to elucidate the antioxidative function.     

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.     

Catalytic spectrofluorimetric determination of superoxide anion radical and superoxide dismutase activity using N,N-dimethylaniline as the substrate for horseradish peroxidase (HRP)

The coupled reaction of N,N-dimethylaniline (DMA) with 4-aminoantipyrine (4-AAP) using superoxide anion radical (O2-) as oxidizing agent under the catalysis of horseradish peroxidase (HRP) was studied. Based on the reaction, O2- produced by irradiating Vitamin B2, (VB2) was spectrophotometricly determined at 554 nm. The linear range of this method was 1.8 x 10(-6)-1.2 x 10(-4) mol l(-1) with a detection limit of 5.3 x 10(-7) mol l(-1). The effect of interferences on the determination of O2- was investigated. The proposed method was successfully applied to the determination of superoxide dismutase (SOD) activity in human blood and mouse blood.     

Determination of the activity of superoxide dismutase using a glassy carbon electrode modified with ferrocene imidazolium salts and hydroxy-functionalized graphene

Microchimica Acta - A glassy carbon electrode was modified with a composite consisting of ferrocene imidazolium salts and hydroxy-functionalized graphene in a Nafion matrix. The electrode is shown...     

Simple and rapid catalytic spectrophotometric determination of superoxide anion radical and superoxide dismutase activity in natural medical vegetables using phenol as the substrate for horseradish peroxidase

The coupling reaction of 4-aminoantipyrine (4-AAP) with phenol using the superoxide anion radical ( ) as oxidizing agent under the catalysis of horseradish peroxidase (HRP) was studied. Based on the reaction, produced by irradiating vitamin B₂ (V_B2) was spectrophotometrically determined at 510 nm. Under the optimum experimental conditions, the relationship between A ₅₁₀ and concentration was linear in the range 9.14×10^–6–1.2×10^–4 mol L^–1. The detection limit was determined to be 1.37×10^–6 mol L^–1. A possible reaction mechanism was discussed. The effect of interferences and surfactants on the determination of was also investigated. The proposed method was applied to determine superoxide dismutase activity in garlic, scallion, and onion with satisfactory results.     

Modified expression of plasma glutathione peroxidase and manganese superoxide dismutase in human renal cell carcinoma

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate thousands of polypeptides and to highlight the modification of protein expression in malignant diseases. By applying 2-D PAGE to ten normal human kidney and ten homologous renal cell carcinoma (RCC) tissues, we found two peptides in all ten normal tissues but not in RCCs and, conversely, two peptides were detected in all RCCs but not in normal tissues. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and internal sequence analysis, the two first peptides were identified as two isoforms of plasma glutathione peroxidase (GPxP). The two other peptides isolated in all RCCs but not in normal tissues were identified by N-terminal sequence analysis as multimeric forms of manganese superoxide dismutase (Mn-SOD). No multimeric Mn-SODs and only two monomeric forms were detected in normal tissues. GPxP and Mn-SOD are metallo-enzymes encoded on chromosome 5q32 and on chromosome 6p25, respectively. Their regions are within the locus 5q21-->qter and 6q21-6q27 on which deletions and translocations are described in some cytogenetic studies of RCC transformation. Therefore, our results might suggest a correlation between the modified expression of GPxP and Mn-SOD in tumor tissues and chromosomal modifications, and that the two proteins may be putative markers for diagnosis of RCC.     

Unexpected superoxide dismutase antioxidant activity of ferric chloride in acetonitrile

The azobis(isobutyronitrile)-initiated autoxidation of gamma-terpinene in acetonitrile at 50 degrees C yields only p-cymene and hydrogen peroxide (1:1) in a chain reaction carried by the hydroperoxyl radical, HOO. (Foti, M. C.; Ingold, K. U. J. Agric. Food Chem. 2003, 51, 2758-2765). This reaction is retarded by very low (microM) concentrations of FeCl(3) and CuCl(2). The kinetics of the FeCl(3)-inhibited autoxidation are consistent with chain-termination via the following: Fe(3+) + HOO. <==>[Fe(IV)-OOH](3+) and [Fe(IV)-OOH](3+) + HOO. --> Fe(3+) + H2O2 + O2. Thus, FeCl(3) in acetonitrile can be regarded as a very effective (and very simple) superoxide dismutase. The kinetics of the CuCl(2)-inhibited autoxidation indicate that chain transfer occurs and becomes more and more important as the reaction proceeds, i.e., the inhibition is replaced by autocatalysis. These kinetics are consistent withreduction of Cu2+ to Cu+ by HOO. and then the reoxidation of Cu+ to Cu2+ by both HOO.and the H2O2 product. The latter reaction yields HO. radicals which continue the chain.     

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。     

Seven-coordinate iron and manganese complexes with acyclic and rigid pentadentate chelates and their superoxide dismutase activity

The reactions of seven-coordinate [Fe(III)(dapsox)(H(2)O)(2)]ClO(4).H(2)O (1), [Fe(II)(H(2)dapsox)(H(2)O)(2)](NO(3))(2).H(2)O (2), and [Mn(II)(H(2)dapsox)(CH(3)OH)(H(2)O)](ClO4)2(H2O) (3) complexes of the acyclic and rigid pentadentate H(2)dapsox ligand [H2dapsox = 2,6-diacetylpyridinebis(semioxamazide)] with superoxide have been studied spectrophotometrically, electrochemically, and by a submillisecond mixing UV/vis stopped-flow in dimethyl sulfoxide (DMSO). The same studies were performed on the seven-coordinate [Mn(II)(Me(2)[15]pyridinaneN(5))(H(2)O)(2)]Cl(2).H(2)O (4) complex with the flexible macrocyclic Me(2)[15]pyridinaneN(5) ligand (Me(2)[15]pyridinaneN(5) = trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene), which belongs to the class of proven superoxide dismutase (SOD) mimetics. The X-ray crystal structures of 2-4 were determined. All complexes possess pentagonal-bipyramidal geometry with the pentadentate ligand in the equatorial plane and solvent molecules in the axial positions. The stopped-flow experiments in DMSO (0.06% of water) reveal that all four metal complexes catalyze the fast disproportionation of superoxide under the applied experimental conditions, and the catalytic rate constants are found to be (3.7 +/- 0.5) x 10(6), (3.9 +/- 0.5) x 10(6), (1.2 +/- 0.3) x 10(7), and (5.3 +/- 0.8) x 10(6) M(-1) s(-1) for 1-4, respectively. The cytochrome c McCord-Fridovich (McCF) assay in an aqueous solution at pH = 7.8 resulted in the IC(50) values (and corresponding kMcCF constants) for 3 and 4, 0.013 +/- 0.001 microM (1.9 +/- 0.2 x 10(8) M(-1) s(-1)) and 0.024 +/- 0.001 microM (1.1 +/- 0.3 x 10(8) M(-1) s(-1)), respectively. IC(50) values from a nitroblue tetrazolium assay are found to be 6.45 +/- 0.02 and 1.36 +/- 0.03 microM for 1 and 4, respectively. The data have been compared with those obtained by direct stopped-flow measurements and discussed in terms of the side reactions that occur under the conditions of indirect assays.     

Replacement of native copper in superoxide dismutase with copper-64 without much loss in its enzymatic activity

Superoxide dismutase, containing copper and zinc, has been labeled with copper-64 by incubating the prepared apoenzyme with cupric(65) chloride at room temperature. No significant loss in the enzymatic activity was observed after labeling. The incorporation of copper-64 was ascertained by starch gel electrophoresis and high performance liquid chromatography. The labeling efficiency was found to be >95%.     

Synthesis,characterization and superoxide dismutase mimetic activity of ruthenium(III) oxime complexes

Vicinal carbonyl-oxime and oxime-imine ligands were used in the synthesis of new RuIII oxime complexes and the isolated chelates were characterized by elemental analysis, electrical conductance and magnetic moment measurements. I.r., u.v.–vis. and e.s.r. spectroscopic analysis methods were also employed. The spectral data were utilized to compute the important ligand field parameters B, β and Dq. The carbonyl-oxime ligand coordinates through the nitrogen of =N-OH to form a five-membered chelate ring. Replacement of the C=O group by C=N-NH2 induces the =N-OH group to coordinate through the oxygen, forming thereby a six-membered chelate ring. The quadridentate tetraaza ligand (L7) coordinates with RuIII through its nitrogen donors in the equatorial position with loss of one of the oxime protons and concomitant formation of an intramolecular hydrogen bond. The spectral and magnetic results suggest a slightly distorted octahedral environment around the RuIII ion. The superoxide dismutase (SOD) mimetic activity of the prepared complexes was assessed for their ability to inhibit the reduction of nitroblue tetrazolium (NBT). The results demonstrate that most of the complexes have promising SOD-mimetic activity. A probable mechanism for the catalytic scavenging of O2− by RuIII oximes is proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis,characterization and superoxide dismutase activity of some octahedral nickel(II) complexes

Four new mixed ligand nickel(II) complexes viz., [Ni(tren)(phen)](ClO₄)₂ (1), [Ni(tren)(bipy)](ClO₄)₂ (2), [Ni(SAA)(PMDT)] · 2H₂O (3) and [Ni(SAA)(TPTZ)] (4) (tren = tris(2-aminoethylamine), phen = 1,10-phenanthroline, bipy = 2,2′-bipyridine, SAA = salicylidene anthranilic acid, PMDT = N,N,N′,N″,N″-pentamethyldiethylenetriamine, TPTZ = 2,4,6-tri(2-pyridyl)-1,3,5-triazine) have been synthesized and characterized by means of elemental analysis, spectroscopic, magnetic susceptibility and cyclic voltammetric measurements. Single crystal X-ray analysis of [Ni(tren)(phen)](ClO₄)₂ (1) and [Ni(SAA)(PMDT)] · 2H₂O (3) has revealed the presence of a distorted octahedral geometry. Superoxide dismutase activity of these complexes has also been measured.     

Complexation, structure, and superoxide dismutase activity of the imidazolate-bridged dinuclear copper moiety with beta-cyclodextrin and its guanidinium-containing derivative

An imidazolate-bridged homodinuclear complex, {[Cu(L)(H2O)]2(im)}(ClO4)3 (1), assembled with beta-cyclodextrin (betaCD) and its guanidinium-containing derivative (betaGCD), and thus a helical inclusion complex, {[Cu(L)(H2O)(betaCD)]2(im)}(ClO4)3 (2), were successfully isolated and structurally characterized. Structural analysis showed that each Cu(II) ion has a distorted square pyramidal N4Ow coordination sphere and forms a chiral chain through hydrogen-bonding and hydrophobic interactions. The UV-vis data showed that such a chain can provide the imidazolate bridge additional stability and results in the dissociation equilibrium taking place at the physiological pH. The obtained IC50 value for 2 (0.23 muM) showed a high superoxide dismutase (SOD) activity, which corresponds to a highly stable imidazolate bridge. Interestingly, the guanidinium-containing 1/betaGCD system showed higher SOD activity (IC50 = 0.16 muM), which is enhanced at least by 30% in comparison with that of guanidinium-lacking 2. This result supports that the positive guanidinium plays a role in the catalytic mechanism of Cu,Zn-SOD by ensuring that superoxide enters and peroxide leaves rapidly from the coordination sphere of the copper ion.     

Superoxide dismutase activity of iron(II)TPEN complex and its derivatives

Superoxide is involved in the pathogenesis of various diseases, such as inflammation, ischemia-reperfusion injury and carcinogenesis. Superoxide dismutases (SODs) catalyze the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, and can protect living cells against the toxicity of free radicals derived from oxygen. Thus, SODs and their functional mimics have potential value as pharmaceuticals. We have previously reported that Fe(II)tetrakis-N,N,N',N'-(2-pyridylmethyl)ethylenediamine (Fe(II)TPEN) has an excellent SOD activity (IC50 = 0.5 microM) among many iron complexes examined (J. Biol. Chem., 264, 9243-9249 (1989)). Fe(II)TPEN can act like native SOD in living cells, and protect Escherichia coli cells from free radical toxicity caused by paraquat. In order to develop more effective SOD functional mimics, we synthesized Fe(II)TPEN derivatives with electron-donating or electron-withdrawing groups at the 4-position of all pyridines of TPEN, and measured the SOD activities and the redox potentials of these complexes. Fe(II) tetrakis-N,N,N',N'-(4-methoxy-2-pyridylmethyl)ethylenediamine (Fe(II)(4MeO)4TPEN) had the highest SOD activity (IC50 = 0.1 microM) among these iron-based SOD mimics. In addition, a good correlation was found between the redox potential and the SOD activity of 15 Fe(II) complexes, including iron-based SOD mimics reported in the previous paper (J. Organometal. Chem., in press). Iron-based SOD mimics may be clinically applicable, because these complexes are generally tissue-permeable and show low toxicity. Therefore our findings should be significant for the development of clinically useful SOD mimics.