Lipophilic ionophore complexes as superoxide dismutase mimetics

A wide range of metal ion complexes exhibit superoxide dismutase like activities as detected by inhibition of nitroblue tetrazolium reduction. Mn(II) and Cu(II) complexes of EDTA, EHPG, and EGTA exhibit SOD like activities commensurate with many of the purpose-built SOD mimics. Here, we report analogous lipophilic chelators that localise metal ions (Cu(II), Mn(II), and Fe(III)) in the lipid membranes and lipoproteins to protect them from superoxide mediated oxidative damage. Spectroscopic titrations and Jobs method confirm that both 1:1 and 2:1 metal ion monensin complexes form. The cupric complexes are the most active exhibiting IC(50) values of 0.09 and 0.18 microM for 2Cu(II)-monensin and Cu(II)-monensin, respectively, for superoxide destruction. In addition, the IC(50) value for Mn(II)-monensin is 0.31 microM. In conclusion, Mn(II) and Cu(II) complexes of the ionophore monensin exhibit considerable superoxide scavenging activities and represent a novel class of catalytic antioxidants for the protection of lipid structures.     

Macrocyclic copper(II) complexes: superoxide scavenging activity, structural studies and cytotoxicity evaluation

Synthetic superoxide dismutase mimetics have emerged as a potential novel class of drugs for the treatment of oxidative stress related diseases. Among these agents, metal complexes with macrocyclic ligands constitute an important group. In this work we synthesized five macrocyclic copper(II) complexes and evaluated their ability to scavenge the superoxide anions generated by the xanthine-xanthine oxidase system. Two different endpoints were used, the nitro blue tetrazolium (NBT) reduction assay (colorimetric method) and the dihydroethidium (DHE) oxidation assay (fluorimetric method). IC(50) values in the low micromolar range were found in four out of five macrocyclic complexes studied, demonstrating their effective ability to scavenge the superoxide anion. The IC(50) values obtained with the NBT assay for the macrocyclic copper(II) complexes, were consistently higher, approximately threefold, than those obtained with the DHE assay. Spectroscopic and electrochemical studies were performed in order to correlate the structural features of the complexes with their superoxide scavenger activity. Cytotoxicity assays were also performed using the MTT method in V79 mammalian cells and we found that the complexes, in the range of concentrations tested in the superoxide scavenging assays were not considerably toxic. In summary, some of the presented macrocyclic copper(II) complexes, specially those with a high stability constant and low IC(50), appear to be promising superoxide scavenger agents, and should be considered for further biological assays.     

Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes

Redox-active metal ions such as Fe(II)\(III) and Cu(I)\(II) have been proposed to activate reactive oxygen and nitrogen species (RONS) and thus, perpetuate oxidative damage. Here, we show that concentrations of metal ions and EDTA complexes with superoxide-destroying activities equivalent to 1 U SOD are Fe(III) 5.1 microM, Mn(II) 0.77 microM, Cu(II)-EDTA 3.55 microM, Fe(III)-EDTA 2.34 microM, and Mn(II)-EDTA 1.38 microM. The most active being the aquated Cu(II) species which exhibited superoxide-destroying activity equivalent to 2U of SOD at 0.29 microM. Hydrogen peroxide-destroying activities were as follows Fe(III)-EDTA ca. 70 U/mg and aquated Fe(III) 141 U/mg. In contrast, DTPA prevented superoxide-destroying activity and significantly depleted hydrogen peroxide-destroying activity. In conclusion, non-protein bound transition metal ions may have significant anti-oxidant effects in biological systems. Caution should be employed in bioassays when chelating metal ions. Our results demonstrate that DTPA is preferential to EDTA for inactivating redox-active metal ions in bioassays.     

Determination of superoxide dismutase-like activity in some divalent metal saccharinates

The superoxide-dismutase-like activity of a series of divalent metal saccharinates of general stoichiometry [M^II(Sac)₂(H₂O)₄]·2H₂O (with M^II=Mn,Fe,Co,Ni,Cu,Zn) has been investigated using the nitroblue tetrazolium O ₂ ⁻ reduction assay. The results show that all these complexes possess the capability to dismutate the superoxide anion generated in the xanthine/xanthine oxidase system. Interestingly, the greatest activity is shown by the corresponding copper complex. The results are discussed and compared with those obtained for native superoxide dismutase, which was tested under the same experimental conditions. Dedicated to Prof. Pedro J. Aymonino on the occasion of his 65^th birthday.     

Experimental evidence that flavonoid metal complexes may act as mimics of superoxide dismutase

Radical scavenging activities of flavonoids rutin, taxifolin, (-)-epicatechin, luteolin, and their complexes with transition metal (Fe2+, Fe3+, and Cu2+) towards superoxide were determined using illumination of riboflavin as source and NBT as detector of O*2-. The scavenger potencies of flavonoid metal complexes were significantly higher than those of the parent flavonoids. To elucidate the mechanism of this phenomenon, the rates of superoxide-dependent oxidation of flavonoids and their metal complexes in photochemical system with riboflavin were examined. It was found for the first time that flavonoids bound to metal ions were much less subjected to oxidation compared with those of free compounds. The findings directly demonstrate superoxide scavenging activity of metal ions in complexes with flavonoids and support earlier suggestions that flavonoid metal complexes may exhibit superoxide dismuting activity.     

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller, J. Am. Chem. Soc. 120(3) (1998) 461–467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn²⁺, Fe³⁺ or Fe²⁺ are bound, based on EPR and NMR spectroscopy. However, the mutant’s Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD’s activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn²⁺, Fe³⁺ and ¹H in the presence of Fe²⁺ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe²⁺SOD and Fe²⁺(Mn)SOD by ¹⁵N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe²⁺ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower E_m in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH⁻ coordinated to the metal ion.     

Copper(II) complexes as superoxide dismutase mimics: Synthesis, characterization, crystal structure and bioactivity of copper(II) complexes

Two new copper(II) complexes of the type [Cu(L)X₂), where L = (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide X = Cl/Br have been synthesized and characterized by elemental analyses, FAB (fast atomic bombardment) magnetic measurements, electronic absorption, conductivity measurements cyclic voltammetry (CV) and Electron paramagnetic resonance (epr) spectroscopy. The structures of these complexes determined by single crystal X-ray crystallography show a distorted square based pyramidal (DSBP) geometry around copper(II) metal center. The distorted CuN₂OX (X = Cl/Br) basal plane in them is comprised of two nitrogen and one oxygen atoms of the meridionally coordinated ligand and a chloride or bromide ion and axial position is occupied by other halide ion. The epr spectra of these complexes in frozen solutions of DMSO showed a signal at g ca. 2. The trend in g-value (g_|| > g_⊥ > 2.00) suggest that the unpaired electron on copper(II) has d_x²-y² character. Biological activities in terms of superoxide dismutase (SOD) and antimicrobial properties of copper(II) complexes have also been measured. The superoxide dismutase activity reveals that these two complexes catalyze the fast disproportionation of superoxide in DMSO solution.     

Permeability of iodide in multilamellar liposomes modeled by two compartments and a reservoir

A previously published rate law for the diffusion of iodide from multilamellar egg phosphatidylcholine liposomes (Schullery, S.E. (1975) Chem. Phys. Lipids 49–58) is fitted to the relatively simple mathematical model of two compartments in series with a reservoir. All of the inner liposome compartments are assumed to behave as effectively one compartment in series with the liposome's outermost compartment. Based on this model, reasonable values are calculated for the fraction of the total solution trapped by liposomes which is in the outermost liposome compartment, 17%, and the permeability coefficient of iodide against isotonic, mixed iodide-chloride solution, $\text{2 · 10}{}^{\mathrm{?9}}\text{cm/s}$.     

Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle

Fast (extensor digitorum longus) and slow (soleus) rat skeletal muscles served as the source for isolation and biochemical comparison of two distinct surface membrane fractions with properties of the sarcolemma and transverse tubular system. Enriched sarcolemmal membrane from soleus demonstrated a lighter density after sucrose density centrifugation. Sialic acid content was 1.5-fold higher in soleus (62 nmol/mg) than extensor (40 nmol/mg). The specific activity of (Na⁺ + K⁺ + Mg²⁺)-ATPase was similar (1.40 and 1.65 μmol P_i/mg per 5 min) with the soleus enzyme displaying a (1) greater resistance to inhibition by ouabain, and (2) broader ionic ratio ($\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$) requirement than extensor enzyme. The polypeptide and phospholipid composition showed no major differences between the two muscle types.The second surface membrane fraction, tentatively identified as transverse tubule, differed in membrane composition. The major polypeptide of extensor was of 95 000 molecular weight whereas for soleus a M_r = 28 000 species was dominant. Total phospholipid content of soleus was 1.5-fold greater than extensor due mostly to increased levels of phosphatidylcholine and phosphatidylethanolamine. Endogenous membrane protein kinase for the 28 000 molecular weight polypeptide was found exclusively in this membrane. The reaction conditions were identical for extensor and soleus since both required divalent cations (Ca²⁺ and Mg²⁺) and neither was affected by cyclic AMP. Soleus showed a 2-fold higher capacity for phosphate incorporation than extensor.These studies show that surface membrane fractions derived from fast and slow muscles differ in terms of functional and compositional properties. These differences are specific not only for the surface membrane but for the muscle type and may relate to the known physiological differences observed between fast and slow mammalian muscle.     

超氧化物岐化酶在医学领域的研究现状

超氧化物歧化酶(SOD)是生物体内清除超氧阴离子自由基(O2-.)的一种重要金属酶,具有重要的生理功能,在医药、食品等方面有广泛的应用前景,是目前医学、分子生物学领域研究的热点之一。现对SOD的分类、分布、结构、性质、在相关疾病中的研究进展、临床应用等方面做一综述。     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Inhibition of Neuronal Apoptosis by a Metalloporphyrin Superoxide Dismutase Mimic

Abstract: The objective of this study was to determine whether free radicals play a pathogenic role in neuronal apoptosis. The ability of Mn(III) tetrakis(benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimic, to inhibit staurosporine-induced neuronal apoptosis was tested in mixed cerebrocortical cultures. Staurosporine produced concentration-dependent cell death that was markedly inhibited by MnTBAP. Immunocytochemical analyses of cultures for neuron- and astrocyte-specific markers revealed that high concentrations of staurosporine induced the death of both neurons and astrocytes; both cell types were protected by MnTBAP. A less active congener of MnTBAP failed to protect cells against staurosporine-induced apoptosis. MnTBAP also protected cortical cultures against ceramide-induced apoptosis. These results support a role for oxidative stress in neuronal apoptosis.     

Expression of the Cu,Zn superoxide dismutase of Aspergillus fumigatus as determined by immunochemistry and immunoelectron microscopy

Abstract A polyclonal antibody against purified Cu,Zn superoxide dismutase (SOD) from the pathogen Aspergillus fumigatus was raised in a sheep. This antibody recognised purified A. fumigatus SOD, together with a single band of 19 kDa in A. fumigatus cytoplasmic antigen, by immunodevelopment of Western blots. The polyclonal serum did not recognise either the manganese or iron containing forms of the enzyme; however, it was reactive against putative Cu,Zn SODs in other members of the genus Aspergillus . Immunofluorescent staining of A. fumigatus cultures demonstrated expression of the Cu,Zn SOD in conidia and hyphae, with the cell wall staining particularly intensely. Conidiophores were stained in an uniformly intense pattern. Immunoelectron microscopy confirmed that the SOD was present within the hyphal cell wall, although there was also labelling in the cytoplasm. SOD may protect Aspergillus against oxidants produced by immune effector cells and these observations demonstrate that the enzyme is available to perform its antioxidant function within the cell wall.     

MnSOD expression inhibited by electromagnetic pulse radiation in the rat testis

Male Sprague Dawley rats were exposed to EMP irradiation of 100 kV/m peak-to-peak e-field intensity and different numbers of pulses. Rat sperm samples were prepared for analysis of sperm qualities; Testes were assessed by transmission electron microscopy and serum hormone concentrations were examined by radioimmunoassay; Enzymatic activities of Total-superoxide dismutase(T-SOD) and manganese-superoxide dismutase (MnSOD), the mRNA levels of MnSOD and cuprozinc-superoxide dismutase (CuZnSOD), and the density of malondialdehyde (MDA) were also determined. EMP irradiation did not affect spermatozoon morphology, micronucleus formation rate, sperm number or viability, but the acrosin reaction rate decreased at 24 h and 48 h and recovered by 72 h after irradiation as compared to the controls. The ultrastructure of rat testis displayed more serious damage at 24 h than at other time points (6 h, 12 h, 48 h). Serum levels of luteotrophic hormone (LH) and testosterone (T) were elevated in irradiated rats as compared to controls. After irradiation, enzymatic activities of T-SOD and MnSOD were reduced by 24 h, consistent with the changes observed in MnSOD mRNA expression; MDA content increased at 6 h in turn. These studies have quantified the morphological damage and dysfunction in the rat reproductive system induced by EMP. The mechanism of EMP induced damage may be associated with the inhibition of MnSOD expression.     

Photosynthetic apparatus of chilling-sensitive plants. XI. Reversibility by light of cold- and dark-induced inactivation of cyanide-sensitive superoxide dismutase activity in tomato leaf chloroplasts

(1) The inactivation of cyanide-sensitive, copper- and zinc-containing superoxide dismutase activity in chloroplasts following cold and dark storage of both detached leaves and growing tomato plants is accompanied by a decrease in copper and zinc content in both chloroplast preparations and butanol extracts of the enzyme. In contrast, this treatment of chloroplast preparations affects neither superoxide dismutase activity nor copper and zinc content. (2) Copper- and zinc-containing superoxide dismutase is not reactivated following the 2–3 h illumination of cold- and dark-stored detached leaves. However, prolonged illumination of growing seedlings results in the restoration of both the enzyme activity and copper and zinc content in chloroplasts. (3) The data suggest that the dissociation of copper, and probably of zinc, from the enzyme during cold and dark treatment of either detached leaves or growing plants and reincorporation of the metals following the illumination of intact plants are responsible for the reversible inactivation of chloroplast cyanide-sensitive superoxide dismutase of chilling-sensitive plants.     

Free radical scavenging activity of the Japanese herbal medicine Toki-Shakuyaku-San (TJ-23) and its effect on superoxide dismutase activity,lipid peroxides,glutamate, and monoamine metabolites in aged rat brain

The free radical scavenging activity of the Japanese herbal medicine, Toki-Shakuyaku-San (TJ-23; TSUMURA & Co., Tokyo, Japan), was examined using electron spin resonance (ESR) spectrometry. TJ-23 scavenged 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), superoxide (O₂ ⁻), and hydroxyl radicals (·OH) dose-dependently. It also diminished carbon centered radicals (·C) generated by oxidative stress and inhibited thiobarbituric acid-reactive substances (TBARS) formation in mouse cortex homogenate. In addition, the effect of TJ-23 on the concentration of neurotransmitters and TBARS formation, and superoxide dismutase (SOD) activity in the cortex, hippocampus and striatum of the aged rat brain was studied. The concentrations of the metabolites of monoamines, glutamate and glutamine were decreased by 4 weeks of oral administration of TJ-23. The SOD activity of mitochondrial fraction was increased and TBARS formation was significantly suppressed. These results suggest that TJ-23 has an antioxidant action and would have a prophylactic effect against free radical-mediated neurological diseases associated with aging.     

Trypanosoma cruzi: effect of benznidazole therapy combined with the iron chelator desferrioxamine in infected mice

Iron chelators have been employed in various studies aimed at evaluating the relationship between the iron status of the host and the development of infection. In the present study, the effects of benznidazole (BZ) therapy in combination with the iron chelator desferrioxamine (DFO) on the development of infection in mice inoculated with Trypanosoma cruzi Y strain have been investigated. Infected mice treated with DFO presented lower levels of parasitemia compared with infected untreated animals. Therapy with BZ for 21 days, with or without DFO, led to decreased parasitemia and reduced mortality, but BZ in combination with DFO treatment for 35 days (BZ/DFO-35) gave 0% mortality. All infected groups presented lower levels of iron in the liver, but serum iron concentrations were greater in DFO-35 and BZ/DFO-35, whereas hemoglobin levels were higher in BZ/DFO-35 and lower in DFO-35 compared with other treated groups. The percentage cure, determined from negative hemoculture and PCR results in animals that had survived for 60 days post-infection, was 18% for BZ and BZ/DFO-35, 42% for BZ combined with DFO for 21 days, and 67% for DFO-35. The results demonstrate that modification in iron stores increases BZ efficacy.     

Selective 15N labeling and direct observation by NMR of the active-site glutamine of Fe-containing superoxide dismutase

The glutamine in position 69 is one of only three conserved active-site amino acid differencesbetween Fe- and Mn-containing superoxide dismutases (SODs). We have refined theconditions for extremely selective labeling of the side chains of glutamine with 15N, and thusobtained dramatically simplified spectra, despite the large size of Fe-SOD. The improvedresolution afforded by such highly specific labeling permits the use of direct 15N detectionto observe and assign Gln 69, even though its distance to the paramagnetic Fe2+ is only 5Å. Selective glutamine side-chain labeling is inexpensive and has general utility forlarge (and paramagnet-containing) proteins.