Superoxide dismutase,

Superoxide dismutase (SOD EC 1.15.1.1) is the term used for a number of metalloproteins that catalyse the following reaction  

Assay of SOD is difficult because of the free radical nature of its substrate (02 ) which must of necessity be generated within the assay system and which cannot be measured directly by simple analytical 

Reagents 50 mM potassium phosphate buffer, pH 7.8, containing 0.1 mM ethylene diamine tetraacetic acid, 0.76 mg xanthine in 10 ml 0.001 M sodium hydroxide solution, 24.8 mg cytochrome c in 100 ml phosphate buffer, pH 7.8. 

Solution A is prepared by mixing 10 ml of the xanthine solution with 100 ml of the cytochrome c solution the mixture is stable at 4°C for about 3 days. 

Solution B is a solution of xanthine oxidase (Grade I, Sigma) in the EDTA-containing buffer, pH 7.8, to give an activity of about 0.2 U/ 

Superoxide dismutase (SOD) catalyzes the reaction shown in Equation (1)  

It is believed that MnSOD plays a pivotal role in many diseases. There have been many reviews of the biochemistry of MnSOD and focusing on the structural aspects of the enzyme. Four different types of SOD are known, a Cu/Zn-containing SOD, a FeSOD, a NiSOD, and MnSOD. MnSODs, which are structurally related to the FeSODs, have a of 23,000 ( 200 amino acids) and function as a dimer or as a tetramer. MnSOD catalyzes the dismutation reaction by cycling between the - -2 and +3 oxidation states. One proton is taken up by the system in each step (Equation (2))  

Crystal structure determinations of MnSODs from organisms ranging from E. coli to humans have been reported. Structural determinations of note include those by Jameson et al. on the E. coli enzyme and mutant forms of this enzyme with atomic resolution,a cambialistic superoxide dismutase from Porphyromonas gingivalis, and mutant forms of the human enzyme the Q143N, and Q143A mutants.The coordination sphere of the 

One area of research interest has been the metal ion specificity of the MnSOD and FeSOD molecules. The tertiary structures of these molecules are very similar and the ligands coordinated to the metal ions are identical. Many organisms contain both forms of the enzyme and each form has an absolute specificity for its metal ion, the enzyme is completely inactive if the wrong metal ion is present. Cambialistic enzymes that occur in some organisms are active with either metal ion present in the active site. Comparisons of the structures of the MnSOD, FeSOD, and the cambialistic enzymes have not revealed any single obvious structural differences that could explain this phenomenon.  

Vance and Miller et al. have shown that the inactivity of enzyme is due to changes in the redox potentials of the enzyme. In order to dismute 02 the redox potential of the enzyme must lie between the E° values for the reactions shown in Equation (3). The E° value of the E. coli MnSOD enzyme is 0.290 V and that for the FeSOD is 0.220 V. The Fe-substituted form of the Mn enzyme has F ° =—0.240 V and Mn-substituted FeSOD has ii° 0.960V. These values are outside the required range and the changes in redox potentials are not due to changes in the metal ligands. Mutations of His-30 and Tyr-34, two conserved residues in the immediate vicinity of the metal binding site, do not alter the redox potential of the enzyme either  

Superoxide dismutase enzymes are functional dimers of molecular weight (Mr) of approximately 32 kDa. The enzymes contain one copper ion and one zinc ion per subunit. Superoxide dismutase (SOD) metalloenzymes function to disproportionate the biologically harmful superoxide ion-radical according to the following reaction  

One product of this reaction, H2O2, is also a potentially harmful substance. Hydrogen peroxide is removed by the heme iron metalloenzyme catalase according to the following equation  

Electron paramagnetic resonance (EPR) spectra are also discussed by Valentine et al. in reference 21. The splitting of the gy resonance is due to hyperfine coupling between the unpaired electron on Cu(II) and the nuclear spin of the copper nucleus 

The authors find that the CuZnSOD protein backbone (the so-called SOD rack ) remains essentially unchanged in all structures investigated here and therefore assume that backbone changes do not play a role in the catalytic cycle. 

The authors conclude that superoxide ion probably binds in a similar fashion to the azide and that conserved water ligands in the enzyme structure both hydrogen-bond with and help guide the substrates toward the copper ion. If this is the case, then superoxide binds directly to Cu(II) (inner-sphere electron transfer) in the following reaction  

Superoxide dismutase (SOD), an indigenous enzyme in milk, was discussed in section 8.2.10. A low level of exogenous SOD, coupled with catalase, was shown to be a very effective inhibitor of lipid oxidation in dairy products. It has been suggested that SOD may be particularly useful in preserving the flavour of long-life UHT milk which is prone to lipid oxidation. Obviously, the commercial feasibility of using SOD as an antioxidant depends on cost, particularly vis-d-vis chemical methods, if permitted. 

Superoxide Dismutase.—Rate constants for the reactions between Oj and the chelates of salicylate, acetylsalicylate, p-aminosalicylate, and di-isopropyl-salicylate at pH 7.5 range from 0.8 to 2.4x 10 M s compared with native cuprein-copper for which A =1.3x 10 M s per g atom of Cu. Thus, these chelates act as perfect model superoxide dismutases. 

Mechanisms of Complex Formation and Ligand Exchange , in Co-ordination Chemistry , 

Palmer and H. Kelm, Inorg. Chim. Acta, 1978, 29, L278. 

Heremans, Fast Reactions in Solution under High Pressure , in High Pressure Chemistry ed. H. Kelm, Reidel, Dordrecht, 1978 Abstracts of the Second International Symposium on the Mechanisms of Reactions in Solution, University of Kent at Canterbury, 1979. 

In Volume 6 of this series, evidence was summarized for a relatively new mechanism involving covalent hydrate and pseudo-base formation in hydrolysis reactions of complexes of bipy, phen, and terpy. Attack by HjO or OH ion usually occurs at the carbon atoms adjacent to the co-ordinated V-donors (e.g. the 2,9-positions of phen), or at the 4,7-positions of phen, and at similar positions for bipy and terpy. However, for 5-nitro-l,10-phenanthroline, n.m.r. evidence has been found for attack by MeO ion at the 6-position of free and co-ordinated 

Human serum and saliva contain superoxide dismutase (SOD), peroxidase and catalase—antioxidant enzymes that destroy H2O2 and 02 and represent a form of the antioxidant defense against mutagenic factors (Nishioka and Ninoshiba, 1986). Clinical investigations have shown that SOD administration has a significant beneficial effect in cardiac feilure, in which the heart muscle is injured (Fass, 1987). SOD has prospects for application not only in medicine, but also in food industry, where, in combination with catalase and peroxidase, it may be used to prevent oxidation of lipids and other valuable components of food (Taylor and Richardson, 1979). The SOD isolated from certain marine bacteria could be used (Mickelson, 1977) to prevent autooxidation in several test systems. 

SOD and other antioxidant enzymes are produced commercially on a limited scale, mainly for laboratory purposes, being isolated from red blood cells. Propionibacteria are a good source of SOD and their value in this respect rises due to the possibility of multi-purpose processing of the biomass. We have developed (Kraeva and Vorobjeva, 1981a, b) a simple method of isolation and purification of SOD to apparent homogeneity, which is shown in Table 7.5. Since SOD is a thermostable protein, heat treatment was used in the purification, thus significantly reducing the number of purification steps. In the course of purification no enzyme modification was 

Cell-free extract Ammonium sulfate (50-80% saturation) precipitate Heat treatment (70 C, 5 min) Chromatography on DEAE-52 Preparative gel-electrophoresis and re-chromatography on DEAE-52 

Since propionibacteria are approved for use in food industry, die following simple preparation can be recommended for this purpose. After the extraction of vitamin Bn with 20% ethanol, the biomass is disintegrated and cell debris removed by filtration the filtrate contains catalase, peroxidase and SOD activities and can be used as an antioxidant preparation without further purification. 

Oxygen-utilizing organisms have generally evolved specific enzyme-mediated systems that serve to protect the cell from such reactive species. These enzymes include superoxide dismutase (SOD) and catalase or glutathione peroxidase (GSH-px), which catalyse the following reactions  

SOD isolated from bovine liver or erythrocytes has been used medically as an antiinflammatory agent. Human SOD has also been expressed in several recombinant systems, and is currently being evaluated to assess its ability to prevent tissue damage induced by exposure to excessively oxygen-rich blood. 

Debridement refers to the process of cleaning a wound by removal of foreign material and dead tissue. Cleansing of the wound facilitates rapid healing and minimizes the risk of infection due to the presence of bacteria at the wound surface. The formation of a clot, followed by a scab, on a 

The value of proteases in cleansing tissue wounds have been appreciated for several hundred years. Wounds were sometimes cleansed in the past by application of protease-containing maggot saliva. Nowadays, this is usually more acceptably achieved by topical application of the enzyme to the wound surface. In some cases, the enzyme is formulated in an aqueous-based cream, while in others, it is impregnated into special bandages. Trypsin, papain, collagenase and various microbial enzymes have been used in this regard. 

Papain is a cysteine protease isolated from the latex of the immature fruit and leaves of the plant, Carica papaya. It consists of a single 23.4 kDa, 212 amino acid polypeptide and the purified enzyme exhibits broad proteolytic activity. Although it can be used as a debriding agent, it is also used for a variety of other industrial processes, including meat tenderizing, and for the clarification of beverages. 

CH12 RECOMBINANT BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 

Department of Food Science and Technology, Minami- Kyushu University, Takanabe, Miyazaki 884, Japan The Research Institute for Food Science, Kyoto University, 

Superoxide dismutase (SOD) is a key enzyme which constitutes the first line of defense against oxygen toxicity and catalyzes the disproportionation of the superoxide anions to dioxygen and hydrogen peroxide2  

By lowering the steady state concentration of superoxide, SOD protects cells from harmful effects of O2 itself and of other reactive oxygen species derived from OL3-5  

Nevertheless, with respect to the catalysis of the dismutation of O2, little difference has been found among the three types of SOD.I0) The reaction rate constants between O2 and the enzyme are about 2X109 M- s at neutral pH. However, although the catalytic rate of the CuZn-SOD is constant between pH 5 and 9.5, those of the Fe- and Mn-SODs become progressively lower as the pH is raised above 8.5. 

Structural information on the three types of SOD have revealed structure-function relationships. In this chapter, we describe the phylogenic distribution, gene regulation and general properties of the three types of SOD. The reaction mechanism of SOD is also discussed based on its structure. 

The stoichiometry of Mn SOD enzymes varies greatly. Whereas the E. coli enzyme is a dimer, SOD enzymes from a number of sources, such as T. thermophilus (8) and chicken liver (14), are tetrameric. Each subunit, however, is consistently about 20 kDa, despite the number of 

While the stoichiometries of the Mn SOD enzymes appear to vary, the properties of the Mn-binding site do not. This is borne out in the electronic spectra of these proteins, which display a great degree of similarity despite the diversity of sources from which they have been isolated (Table II). This type of spectrum is distinctive for manganese in the trivalent oxidation state (3). The native enzymes are EPR silent, as might be anticipated if they contained Mn solely as the trivalent ion (S = 2) (1, 6,12,18-20, 24). However, when the enzymes are denatured, the characteristic six-line pattern of Mn(II) (I = 5/2) appears. Magnetic susceptibility studies with the E. coli SOD were consistent with the presence of a monomeric Mn(III) complex with a zero-field splitting of 1 to 2 cm-1 (4). The enzymes are additionally metal specific (however, see Refs. 36 and 37) metal reconstitution studies with E. coli and B. stearothermophilus revealed a strict requirement for Mn for superoxide dismutase activity (2, 22, 23). 

Preliminary X-ray crystallographic studies have appeared on several Mn SOD enzymes (27-29). The crystal structure of the T. thermophilus enzyme at 4.4 A resolution (27) indicated a high degree of homology between the secondary and tertiary structure of the Mn enzyme and an iron SOD isolated from E. coli (3.1 A resolution) (33). Each possesses a single metal-binding site per subunit. Sequence homologies between the 

The four protein residues which serve as metal ligands are conserved in the amino acid sequences of all Mn SODs completely sequenced to date [i.e., T. thermophilus (44), yeast (45), E. coli (46), human liver (47), and B. stearothermophilus (48). [The DNA sequence of the E. coli Mn SOD gene and mouse Mn SOD gene have also recently been determined (49, 55).] Thus, it seems quite probable that the Mn-binding site structure is analogous (or nearly so) in all Mn SODs. Further support for this comes from EPR studies of the Cu(II)-substituted B. stearothermophilus enzyme, which indicate the presence of three imidazole ligands in a rhombic metal complex (50). 

Other than in prokaryotic cells which lack mitochondria and chloroplasts, manganese superoxide dismutases are apparently restricted to the above two organelles in eukaryotic cells (51, 52) this forms strong support for the symbiotic hypothesis for the origin of mitochondria and chloroplasts (53, 54). Kinetic studies of superoxide dismutation by these enzymes indicate three oxidation states of Mn (presumably divalent, trivalent, and tetravalent) are involved in the catalytic cycle (57, 58). They also show that a Mn-02 complex may conceivably be formed. Well-characterized Mn-dioxygen (i.e., 02,02 , 022 ) adducts are extremely rare, the first structurally characterized example being reported only in 1987 (60). 

A scavenging role for MT in inactivating free radicals to prevent damage. 

MT-mediated movement of copper to subcellular locations to minimize toxic effects of the metal. 

A role for MT in providing copper and/or zinc to SOD or other antioxidant proteins. 

A role for MT in mediating the zinc status of MRTFs important in regulating expression of stress response genes. 

Genetic manipulation to specifically alter MT expression (both up and down) in active MT gene transfection, gene knockout, and antisense nucleic acid expression, combined with exposure to physiological events mediating oxidative stress, will provide further insights into the relationship between MT and oxygen radicals in the future. 

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Klapper, I., Hagstrom, R., Fine, R., Sharp, K., Honig, B. Focusing of electric fields in the active site of cu,zn superoxide dismutase. Proteins Struct. Pune. Genet. 1 (1986) 47-79. 

Klapper 1, R Hagstrom, RFine, K Sharp and B Honig 1986. Focusing of Electric Fields in tire Actir e Sit of CuZn Superoxide Dismutase Effects of Ionic Strength and Amino-Acid Substitution. Proteins Structure, Function and Genetics 1 47-59. 

Super milling dyes Supermumetal Supernovas Superoxide dismutase... 

Erythrocuprein, which contains about 60 wt % of the erythrocyte copper, hepatocuprein, and cerebrocuprein act as superoxide dismutases. Each contains two atoms of copper per molecule, having mol wt ca 34,000. The superoxide ion, O", and peroxide, two main toxic by-products of... 

Copper is one of the twenty-seven elements known to be essential to humans (69—72) (see Mineral nutrients). The daily recommended requirement for humans is 2.5—5.0 mg (73). Copper is probably second only to iron as an oxidation catalyst and oxygen carrier in humans (74). It is present in many proteins, such as hemocyanin [9013-32-3] galactose oxidase [9028-79-9] ceruloplasmin [9031 -37-2] dopamine -hydroxylase, monoamine oxidase [9001-66-5] superoxide dismutase [9054-89-17, and phenolase (75,76). Copper aids in photosynthesis and other oxidative processes in plants. 

Superoxide dismutase has been approved by the FDA for preventing reperfusion injury or damage to donor organ tissue (178). This enzyme is prepared by recombinant DNA technology and marketed by Bristol-Myers and Pharmacia-Chiron. 

Two classes of antioxidants are known the low-molecular weight compounds (tocopherols, ascorbate, -carotene, glutathione, uric acid and etc.) and the proteins (albumin, transferrin, caeruloplasmin, ferritin, etc.) including antioxidant enzymes (e.g. superoxide dismutase, catalase, glutathione peroxidase). 

Copper-zinc-superoxide dismutase (from blood cell haemolysis) [9054-89-1J Mr 32,000... 

CL Eisher, J-L Chen, J Li, D Bashford, L Noodleman. Density-functional and electrostatic calculations for a model of a manganese superoxide dismutase active site in aqueous solution. J Phys Chem 100 13498-13505, 1996. 

J Shen, CF Wong, S Subramaniam, TA Albright, JA McCammon. Partial electrostatic charges for the active center of Cu,Zn superoxide dismutase. J Comput Chem 11 346-350, 1990. 

Figure S.l The enzyme superoxide dismutase (SOD). SOD is a P structure comprising eight antiparallel P strands (a). In addition, SOD has two metal atoms, Cu and Zn (yellow circles), that participate in the catalytic action conversion of a superoxide radical to hydrogen peroxide and oxygen. The eight p strands are arranged around the surface of a barrel, which is viewed along the barrel axis in (b) and perpendicular to this axis in (c). [(a) Adapted from J.S. Richardson. The stmcture of SOD was determined in the laboratory of J.S. and D.R. Richardson, Duke University.)...

McLachlan, A.D. Repeated folding pattern in copper-zinc superoxide dismutase. Nature 285 267-268, 1980. 

Richardson, J.S., et al. Similarity of three-dimensional stmcture between the immunoglobulin domain and the copper, zinc superoxide dismutase subunit. 

Tainer, J.A., et al. Determination and analysis of the 2 A structure of copper, zinc superoxide dismutase. 

When H2O2 is a necessary component of a luminescence system, it can be removed by catalase. If a luminescence system involves superoxide anion, the light emission can be quenched by destroying O2 with superoxide dismutase (SOD). The ATP cofactor usually present in the fresh extracts of the fireflies and the millipede Luminodesmus can be used up by their spontaneous luminescence reactions, eventually resulting in dark (nonluminous) extracts containing a luciferase or photoprotein. The process is, however, accompanied by a corresponding loss in the amount of luciferin or photoprotein. The use of ATPase and the elimination of Mg2+ in the extract may prevent such a loss. 

McCord, J. M., and Fridovich, I. (1969). Superoxide dismutase an enzymic function for erythrocuprein (hemocuprein)./. Biol. Chem. 244 6049-6055. 

Nakano, M. (1990). Assay for superoxide dismutase based on chemiluminescence of luciferin analog. Method. Enzymol. 186 227-232. 

Shimomura, O. (1992). The role of superoxide dismutase in regulating the light emission of luminescent fungi. J. Exp. Botany 43 1519-1525. 

Suzuki, N., etal. (1991). Reaction rates for the chemiluminescence of Cypridina luciferin analogs with superoxide a quenching experiment with superoxide dismutase. Agric. Biol. Chem. 55 157-160. 

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