Isolation and characterization of superoxide dismutase: A personal history and tribute to Joe McCord and Irwin Fridovich

The discovery of superoxide dismutase twenty years ago gave new meaning to work on erythrocuprein. This tribute to the achievement of Joe McCord and Irwin Fridovich is an account of experience of superoxide dismutase from old obscure copper protein of red blood cells to new exciting enzyme of oxygen free-radical metabolism, and an affirmation of the superoxide theory of oxygen toxicity.     

Is the activity-linked electrostatic gradient of bovine Cu, Zn superoxide dismutases conserved in homologous enzymes irrespective of the number and distribution of charges?

Electrostatic potential calculations have been performed on three different Cu, Zn superoxide dismutases (superoxide: superoxide oxidoreductase, EC 1.15 1.1 ), in order to evaluate the degree of conservation of the pattern of the electrostatic interactions between O₂⁻ and the active site recently pointed out in bovine Cu Zn SOD. The three Cu, Zn SODs that have been selected for this study, namely the bovine, ovine, and porcine enzymes, are highly homologous as to reasonably assume identical three-dimensional structure but display large differences in their net charge, as shown by their pI's which span over a wide range pHrange: 8.0 (sheep), 6.5(pig), 5.2(ox). Despite such a large difference in the net protein charge and in the spatial arrangement of electrostatic charges, electrostatic potential calculations show that the electrostatic channel directing the negatively charged substrate toward the positive catalytic site is strictly preserved with the same features for the three proteins. This suggests that the electrostatic funnel for conducting small anions into the active site is a highly conservative property in the evolution of Cu, Zn SOD.     

Involvement of superoxide radicals in the mouse two-cell block.

The effect of oxygen toxicity on the development of mammalian embryos was assessed by the use of superoxide dismutase (SOD), a potent scavenger of superoxide radicals. Mouse pronuclear embryos recovered 17 h after human chorionic gonadotropin (hCG) were cultured in medium BWW at 37 degrees C under an atmosphere of 5% CO2 in air. Culture of mouse pronuclear embryos in the presence of Cu.Zn-SOD (500 micrograms/ml) significantly increased the blastulation rate (44.6%) when compared with the control culture system (4.2%). Essentially the same effects were observed in SOD containing either Mn or Fe in the catalytic center. Heat treatment of the SOD preparation, and the addition of anti-SOD antibodies to the culture medium, significantly reduced the attenuation of the two-cell block by SOD, indicating that this effect is SOD dependent. SOD activity was detected in rabbit oviduct fluid (3.675 +/- 3.084 mIU/mg protein) by electron spin resonance. These results suggest that active oxygen is involved in the two-cell block phenomenon in mouse embryos exposed to air and that SOD in the oviduct may play an important role in the protection of embryos from superoxide radicals.     

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.     

Microplate quantification of plant leaf superoxide dismutases

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals in a broad range of organisms, including plants. Quantification of SOD activity in crude plant extracts has been problematic due to the presence of compounds that interfere with the dose-response of the assay. Although strategies exist to partially purify SODs from plant extracts, the requirement for purification limits the rapidity and practical number of assays that can be conducted. In this article, we describe modification of a procedure using o-dianisidine as substrate that permits relatively rapid quantification of SOD activity in crude leaf extracts in a microplate format. The method employs the use of a commercial apparatus that permits lysis of 12 tissue samples at once and the use of Pipes buffer to reduce interference from compounds present in crude leaf extracts. The assay provided a linear response from 1 to 50 units of SOD. The utility of the assay was demonstrated using tissue extracts prepared from a group of taxonomically diverse plants. Reaction rates with tissue extracts from two grasses were linear for at least 60 min. Tissues of certain species contained interfering compounds, most of which could be removed by ultrafiltration. The presence of plant catalases, peroxidases, and ascorbate in physiological quantities did not interfere with the assay. This approach provides a means to quantify SOD activity in relatively large numbers of plant samples provided that the possibility for the presence of interfering compounds is considered. The presence of interfering compounds in certain plant tissues necessitates caution in interpreting the effects of plant stresses on SOD.     

Distribution of superoxide dismutases, oxidases, and NADH peroxidase in various streptococci

Abstract Managanese-containing superoxide dismutase and NADH oxidase could be detected in all of the 23 strains belonging to different species or serotypes of the genus Streptococcus . NADH peroxidase activity was found in 7 strains. Pyruvate oxidase activity was only detectable in Streptococcus faecalis .     

Periplasmic superoxide dismutases in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 cells cultured microaerobically (dissolved O₂ tension 1% of saturation), expressed proteins with superoxide dismutase (SOD) activity. The majority (roughly 95%) of total cell superoxide dismutase activity was located in the cell periplasm with little or no activity in the cell cytoplasm. Irontype SOD (FeSOD) contributed 88% of the total activity activity detected, although a manganese-type SOD (MnSOD) was present in the periplasm as well. Cells cultured at a higher dissolved O₂ tension (10% of saturation) expressed increased activity of the MnSOD relative to that of the FeSOD.     

Protection of Chinese hamster ovary cells from paraquat-mediated cytotoxicity by a low molecular weight mimic of superoxide dismutase (DF-Mn)

Paraquat exerts a cytotoxic effect of Chinese hamster ovary cells in culture via the superoxide radical (O₂⁻. We have described a superoxide dismutase (SOD) mimic based on manganese (DF-Mn) which consists of a one-to-one complex between desferrioxamine B (Desferal) and MnO₂. It is a small molecular weight molecule, easy to prepare and possesses considerable stability. It is now shown to protect mammalian cells from paraquat toxicity. Thus, 20 μM DF-Mn affords up to complete protection against the cytotoxicity of 200 μM paraquat in Chinese hamster ovary cells. Desferrioxamine B or MnO₂ alone gave no protection. MnCl₂ or catalase provided little or no protection against the paraquat, respectively. Equivalent amounts of human Cu-Zn SOD in terms of activity, also provided no protection. Copper diisopropylsalicylate (CuDIPS) provided limited, yet significant, protection, but this is explained in terms other than SOD activity. Finally, at higher concentrations, purified human SOD, exerts a limited toxicity as well as a protective ability against paraquat (similar to DF-Mn) both of which are eliminated upon heat denaturation of the enzyme. It appears that the SOD mimic, DF-Mn, can enter mammalian cells and can protect against the cytotoxic effects of O₂⁻.     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system

Superoxide radicals in high concentrations were generated from alkaline H₂O₂ without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H₂O₂ concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H₂O₂ led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

In vivo real-time measurement of superoxide anion radical with a novel electrochemical sensor

The dynamics of superoxide anion (O₂⁻) in vivo remain to be clarified because no appropriate method exists to directly and continuously monitor and evaluate O₂⁻ in vivo. Here, we establish an in vivo method using a novel electrochemical O₂⁻ sensor. O₂⁻ generated is measured as a current and evaluated as a quantified partial value of electricity (Q_part), which is calculated by integration of the difference between the baseline and the actual reacted current. The accuracy and efficacy of this method were confirmed by dose-dependent O₂⁻ generation in xanthine–xanthine oxidase in vitro in phosphate-buffered saline and human blood. It was then applied to endotoxemic rats in vivo. O₂⁻ current began to increase 1 h after lipopolysaccharide, and Q_part increased significantly for 6 h in endotoxemic rats, in comparison to sham-treated rats. These values were attenuated by superoxide dismutase. The generation and attenuation of O₂⁻ were indirectly confirmed by plasma lipid peroxidation with malondialdehyde, endothelial injury with soluble intercellular adhesion molecule-1, and microcirculatory dysfunction. This is a novel method for measuring O₂⁻ in vivo and could be used to monitor and treat the pathophysiology caused by excessive O₂⁻ generation in animals and humans.     

How superoxide radical damages the cell

Summary Superoxide is considered to be poorly reactive, and cell damage has been attributed to HO· generated via the Haber-Weiss reaction. The function of O₂ ^– in this reaction is only to reduce Fe³⁺ to Fe²⁺. In vivo, however, superoxide could not out-compete cellular reductants such as glutathione, NADPH, and ascorbate, which makes the observed O₂ ^– toxicity rather puzzling. Little attention has been paid to the idea that, irrespective of its poor chemical reactivity, superoxide might be capable of interacting directly with specific intracellular targets; and that even the Haber-Weiss reaction might be a consequence of such direct interactions. This paper summarizes latest data that support the concept of such a mechanism.Abbreviation SOD Superoxide dismutase     

Production of superoxide dismutases from Proteus mirabilis and Proteus vulgaris

Proteus mirabilis and Proteus vulgaris expressed a combination of superoxide dismutase (Sod) activities, which was assigned to FeSod1, FeSod2 and MnSod for P. mirabilis, and FeSod, MnSod and CuZnSod for P. vulgaris. Production of the Sod proteins was dependent on the availability of iron, whether cells were grown under anaerobiosis or aerobiosis and growth phase. Nalidixic acid and chloramphenicol inhibited cell growth and the iron- and dioxygen-dependent production of Sod. These results support the involvement of metal ions and redox status in the production of Proteus Sods.     

Dynamic and biphasic modulation of nitrosation reaction by superoxide dismutases

It has been shown that superoxide dismutase (SOD) can both potentiate and attenuate NO-mediated toxicity. This present study investigated the role of SOD and GSH in a sustained nitrosative and oxidative environment simulated by the nitric oxide (NO) and superoxide (O(2)(.-)) donor, 3-morpholinosydnonimine (SIN-1). We describe, for the first time, that SOD modulates nitrosative chemistry in a dynamic fashion that is both concentration and time-dependent. Specifically, our results show that SOD's effects on nitrosation are biphasic in nature i.e., while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation. However, even those initially inhibitory higher SOD concentrations became pronitrosative over time. In the presence of physiologically relevant levels of GSH, SOD predominantly exhibits a pronitrosative effect, with a complete loss of antinitrosative effects noted at higher levels of GSH. Our findings likely reflect the complex and dynamic nature of SOD interactions with oxidative and nitrosative species.     

A ready-to-use fluorimetric biosensor for superoxide radical using superoxide dismutase and peroxidase immobilized in sol-gel glasses

In this work, a highly sensitive fluorescent biosensor for quantitative superoxide radical detection, based on the coupled reaction superoxide dismutase-peroxidase enzymes and the use of the probe Amplex red, is described. Superoxide anion radical was produced via oxidation of xanthine by xanthine oxidase. Dismutation of superoxide was catalyzed by superoxide dismutase, generating hydrogen peroxide, which reacted stoichiometrically with the nonfluorescent Amplex red, in the presence of peroxidase, yielding the red-fluorescent oxidation product resorufin. The coupled superoxide dismutase-peroxidase system was immobilized in a single sol-gel matrix. The enzymatic activity of the encapsulated superoxide dismutase-peroxidase system was nearly identical to that of one of the soluble enzymes, indicating that sol-gel encapsulation preserved the hierarchy of the enzyme's activity. Specificity and reusability of the encapsulated system for up to four cycles were also demonstrated. The fluorescent biosensor was able to detect concentrations of superoxide as low as 20 nM in phospholipid model membranes composed of saturated or unsaturated phospholipids. These facts make this biosensor a simple, reliable, and highly sensitive method with a potential use in biological systems, food, and drinks.     

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.)

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a pI value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing patterns revealed the induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alflalfa tissues.     

Biosynthesis and regulation of superoxide dismutases

The past two decades have witnessed an explosion in our understanding of oxygen toxicity. The discovery of superoxide dismutases (SODs) (EC.1.15.1.1), which specifically catalyze the dismutation of superoxide radicals (O₂⁻) to hydrogen peroxide (H₂O₂) and oxygen, has indicated that O₂⁻ is a normal and common byproduct of oxygen metabolism. There is an increasing evidence to support the conclusion that superoxide radicals play a major role in cellular injury, mutagenesis, and many diseases. In all cases SODs have been shown to protect the cells against these deleterious effects. Recent advances in molecular biology and the isolation of different SOD genes and SOD c-DNAs have been useful in proving beyond doubt the physiological function of the enzyme. The biosynthesis of SODs, in most biological systems, is under rigorous controls. In general, exposure to increased pO₂, increased intracellular fluxes of O₂⁻, metal ions perturbation, and exposures to several environmental oxidants have been shown to influence the rate of SOD synthesis in both prokaryotic and eukaryotic organisms. Recent developments in the mechanism of regulation of the manganese-containing superoxide dismutase of Escherichia coli will certainly open new research avenues to better understand the regulation of SODs in other organisms.     

Scavenging of superoxide radical by ascorbic acid

Using acetaldehyde and xanthine oxidase as the source of suPeroxide radical, the second order rate constant for the reaction between ascorbic acid and superoxide radical was estimated to be 8.2 X 10⁷ M^-1 s^-1. In rats, the average tissue concentration of ascorbic acid was of the order of 10^-3 M and that of superoxide dismutase was of the order of 10^-6 M. So, taking together both the rate constants and the tissue concentrations, the efficacy of ascorbic acid for scavenging superoxide radical in animal tissues appears to be better than that of suPeroxide dismutase. The significance of ascorbic acid as a scavenger of superoxide radical has been discussed from the point of view of the evolution of ascorbic acid synthesizing capacity of terrestrial vertebrates.     

Trichinella spiralis: identification and purification of superoxide dismutase

A metalloprotein with superoxide dismutase activity was isolated and purified from muscle-stage Trichinella spiralis. The anti-genicity of the purified enzyme was demonstrated by an immunospecific reaction with T. spiralis antiserum in an enzyme-linked immunosorbent assay. In addition to its presence in somatic extracts of T. spiralis, the enzyme was also excreted into culture fluids in which the muscle-stage larvae had been incubated for periods as short as 3 hr and up to 72 hr. The enzyme was characterized as a copper- and zinc-containing, cyanide-sensitive, superoxide dismutase with a molecular weight of 36,000 (estimated by get filtration), consisting of two subunits of 17,000 Mr (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The isoelectric point was 5.6. Muscle-stage T. spiralis contained one molecular form of the enzyme, whereas adult T. spiralis contained two molecular forms. This enzyme may function as an essential defense mechanism against the highly destructive superoxide radical encountered either intracellularly, as a product of biological oxidation, or externally, as a component of the host's immune system.     

Chromatographic and electrophoretic methods for analysis of superoxide dismutases

A brief overview of the family of superoxide dismutase (SOD) enzymes and their biomedical significance is presented. Methodology for the purification and electrophoretic analysis of superoxide dismutases is reviewed and discussed, with emphasis on the specific problems raised by the separation of individual superoxide dismutase isoenzymes. Purification methods and their performance, as reported in the literature, are summarised in table form. Generally used methods for measuring SOD activity in vitro and SOD visualisation after electrophoresis are outlined, particularly those relevant to the monitoring of progress of SOD purification.     

Dual effects of copper-zinc superoxide dismutase

Copper-zinc superoxide dismutase (CuZnSOD) specifically catalyzes the removal of superoxide radicals to protect cellular function against the generation of superoxide-dependent hydroxyl radicals ((.)OH). However, an unexpected observation reveals that denatured CuZnSOD (dCuZnSOD) itself induces (.)OH formation. This dCuZnSOD-dependent (.)OH generation was not inhibited by active CuZnSOD, suggesting that it is a superoxide-independent process. Sodium cyanide, histidine, and N,N'-diethyldithiocarbamate abolished (.)OH generation, implying that Cu may be responsible for dCuZnSOD-induced (.)OH formation. Catalase eliminated ()OH generation, suggesting that hydrogen peroxide may be involved in the mechanism of dCuZnSOD-mediated (.)OH production. Furthermore, nitric oxide ((.)NO) completely inhibited dCuZnSOD-induced (.)OH radical generation, indicating that (.)NO is an important (.)OH radical scavenger. Our results shed new light on the effect of dysfunctional CuZnSOD and suggest that structural disorder of the enzyme may be one of the endogenous pathways of toxic (.)OH formation in biological systems.