The conserved residue tyrosine 34 is essential for maximal activity of iron-superoxide dismutase from Escherichia coli.

We have expressed, purified, and analyzed the iron-containing superoxide dismutase (FeSOD) of Escherichia coli with mutations directed at tyrosine position 34 to introduce phenylalanine (SODY34F), serine (SODY34S), or cysteine (SODY34C). FeSOD and mutant enzymes were purified from SOD-deficient cells using a GST-FeSOD fusion protein intermediate which was subsequently cleaved with thrombin and repurified. Specific activities were measured using the xanthine-xanthine oxidase method and gave 3148 u/mg for wild-type FeSOD. The SODY34S mutation virtually inactivates the enzyme (42 u/mg); mutation to cysteine greatly reduces activity (563 u/mg), but the SODY34F mutant retains nearly 40% of the activity of wild type (1205 u/mg). Fusion protein intermediates were also shown to be active and were demonstrated to protect SOD-deficient E. coli cells from the induced effects of oxidative stress, with growth rates directly proportional to the specific activities of the expressed mutant enzymes. SODY34F exhibited decreased thermal stability, reduced activity at high pH, and a pronounced increase in sensitivity to the inhibitor sodium azide compared with wild-type FeSOD. These results suggest that tyrosine at position 34 is multifunctional and plays a structural role (probably through hydrogen bonding to glutamine at position 69) in maintaining the integrity of the active site, a stabilizing role at high pH, and a steric role in obstructing access to the active site of both substrate and inhibitor molecules.

Properties of Thermus aquaticus beta-NADH oxidase immobilised on various supports.

beta-NADH oxidase purified from Thermus aquaticus was covalently immobilised on various solid supports. The preparations obtained were compared with the soluble enzyme for activity and kinetic properties. Activated glutaryl-PVA was found to be the best support. The immobilised enzyme was less stable at high temperatures than the soluble enzyme. No differences could be detected in the presence of organic solvents.

Purification and characterization of NADH oxidase from the archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus.

The enzyme NADH oxidase (EC 1.6.99.3) has been isolated from the two thermoacidophilic archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus and characterized. In both organisms the enzyme oxidizes specifically beta-NADH in the presence of molecular oxygen and requires the presence of a flavin cofactor, showing a high specificity for FAD. A stoicheiometric amount of hydrogen peroxide to NADH is formed as the end product of the reaction, indicating that both enzymes are two-electron donors. The purified enzymes exhibit quite different molecular properties. S. acidocaldarius NADH oxidase is a monomeric protein with an estimated molecular mass of about 27 kDa, whereas S. solfataricus NADH oxidase is a dimeric protein with a molecular mass of 35 kDa per subunit; S. solfataricus NADH oxidase is purified as an FAD-containing protein, whereas S. acidocaldarius NADH oxidase does not contain a flavin molecule. Furthermore, a comparison of the N-terminal amino acid sequence shows no similarities either between the two proteins or to any other NADH oxidases. Both enzymes are essentially thermophilic. In the temperature range 20-80 degrees C, the energy of activation is almost the same for both activities, suggesting that similar energetic parameters are required. Also both oxidases display a great stability to heat. The half-life of heat inactivation is about 180 min at 90 degrees C for S. acidocaldarius NADH oxidase and 77 min at 98 degrees C for the S. solfataricus enzyme. The activity of the two enzymes is inhibited by urea and guanidine and are regulated very differently by several organic solvents.

Hapten-carrier interactions and their role in the production of monoclonal antibodies against hydrophobic haptens.

The coupling of haptens to carrier proteins is required for the generation of antihapten antibody response. To date, different polypeptides (such as gelatin, albumin, and keyhole limpet hemocyanin) have been used to increase the immune response against haptens. Different methods of conjugation have also been used to increase the specificity of the antibody response. Our results, obtained through an analysis of the antibody response at both the polyclonal and the monoclonal levels, show that carrier proteins specifically modulate the antibody titer, the specificity of the response, the fusion efficiency, and the number of specific clones. Moreover, the affinity constants of both serum and randomly selected hybridomas directed against different haptens have been found to be exclusively modulated by carrier proteins. In addition, the results show that the carrier protein should be selected considering its possible physicochemical interaction with haptens. Data obtained suggested that any carrier is suitable for hydrophilic haptens, while the choice of the carrier for hydrophobic haptens is critical in obtaining a specific immune response. The use of specific carriers allowed the production of highly specific antibodies: even IgMs, obtained by using these carriers, were able to specifically react with hydrophobic haptens; the case of some IgM monoclonal antibodies specifically reacting with protoporphyrin IX is reported.

Escherichia coli iron superoxide dismutase targeted to the mitochondria of yeast cells protects the cells against oxidative stress.

A gene encoding a fusion protein consisting of Escherichia coli iron superoxide dismutase (FeSOD) with the mitochondrial targeting presequence of yeast manganese superoxide dismutase (MnSOD) was cloned and expressed in E. coli and in Saccharomyces cerevisiae DL1Mn- yeast cells deficient in MnSOD. In the yeast cells the fusion protein was imported into the mitochondrial matrix. However, the presequence was not cleaved. In a control set of experiments, the E. coli FeSOD gene without the yeast MnSOD leader sequence was also cloned and expressed in S. cerevisiae DL1Mn- cells. In this case the FeSOD was located in the cytosol and was not imported into the mitochondrial matrix. E. coli FeSOD, with and without the yeast MnSOD presequence, proved to be active in yeast, but, whereas the FeSOD targeted to the mitochondria of yeast cells deficient in MnSOD protected the cells from the toxic effects of oxidative stress, FeSOD without the yeast MnSOD presequence did not protect the yeast cells deficient in MnSOD against oxidative stress.

The manganese superoxide dismutase gene of Caenorhabditis elegans.

We have cloned and sequenced a gene (sod-3) encoding manganese superoxide dismutase from the nematode Caenorhabditis elegans. The protein-coding region spans 943 bp including three intron sequences and encodes a protein of 227 amino acids (M(r) = 26,367) of which the first 33 amino acids are the presumed mitochondrial-targeting signal peptide. The deduced mature manganese-superoxide dismutase has 194 amino acids (M(r) = 22,193).

The copper/zinc superoxide dismutase gene of Caenorhabditis elegans.

The nucleotide and deduced amino acid sequences of the copper/zinc superoxide dismutase gene (sod-1) from the nematode Caenorhabditis elegans has been determined. The protein coding region is interrupted by three intron sequences covering 608 bp in total and encodes a protein of 158 amino acids (M(r) = 16,307).

An electrochemical method for detection of nucleic acid hybridisation.

Preliminary investigations on the detection of nucleic acid hybridisation by direct electrochemical techniques are described. Initial experiments were performed with calf thymus DNA, while as a model system, plasmid DNA (pEMBL) which can be prepared in single stranded forms from each of two original double stranded plasmids (+19 and -19) was used. Electrochemical analyses have been performed using mercury pool, glassy carbon and screen printed carbon working electrodes.

The role of glutathione in protection against DNA damage induced by rifamycin SV and copper(II) ions.

Incubation of calf thymus DNA in the presence of rifamycin SV induces a decrease in the absorbance of DNA at 260 nm. The effect, was found to be proportional to the antibiotic concentration and enhanced by copper(II) ions. In the presence of rifamycin SV and copper(II), a significant increase in thiobarbituric acid-reactive (TBA-reactive) material is also observed. This effect is inhibited to different degrees by the following antioxidants: catalase 77%; thiourea 72%; glutathione (GSH) 62%; ethanol 52%; and DMSO 34%, suggesting that both hydrogen peroxide (H2O2) and hydroxyl radicals (OH.) are involved in DNA damage. Rifamycin SV-copper(II) mixtures were also found to induce the production of peroxidation material from deoxyribose and, in this case, glutathione and ethanol were the most effective antioxidant substrates with inhibition rates of 91% and 88% respectively. Electrophoretic studies show that calf thymus DNA becomes damaged after 20 min. incubation in the presence of both agents together and that the damaged fragments run with migration rates similar to those obtained by the metal chelating agent 1,10-phenanthroline. Normal DNA electrophoretic pattern was found to be preserved by catalase, and GSH at physiological concentrations and by thiourea. No protection is observed in the presence of ethanol or DMSO. The results obtained indicate the involvement of different reactive species in the degradation process of DNA due to rifamycin SV-copper(II) complex and emphasize the role of reduced glutathione as an oxygen free radical scavenger.

Preparation and kinetic studies of immobilised yeast cytochrome c peroxidase.

Yeast cytochrome c peroxidase (CcP) was purified from baker's yeast and immobilised onto a nylon membrane. The kinetics of the soluble and immobilised forms of the enzyme were investigated for the catalysed oxidation of potassium ferrocyanide in the presence of H2O2 and m-chloroperoxybenzoic acid. The pH dependence of the two forms of the enzyme differed. Although both the soluble and the immobilised enzymes showed optimal activity at pH 6.2, a different kinetic behaviour was demonstrated. Both forms of the enzyme showed similar activity toward H2O2, although when m-chloroperoxybenzoic acid was replaced as the electron acceptor, the immobilised form of the enzyme had a reduced turnover number and an increased Km. The activation energy of immobilised CcP was greater in the presence of both H2O2 [16.6 kJ mol-1] and m-chloroperoxybenzoic acid [37.9 kJ mol-1] than for soluble CcP [11.4 and 23.4 kJ mol-1, respectively]. The activities of both soluble and immobilised CcP were greatly reduced above 45 degrees C, although at higher temperatures the immobilised enzyme retained a relatively greater percentage of its maximum activity.

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes.

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.

Polymers of Cu/Zn superoxide dismutase produced by cross-linking with glutaraldehyde.

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer, with a molecular weight of 64,000 has been purified by gel filtration. The functional properties of the tetramer have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimeric enzyme. The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be different with the tetramer accumulating predominantly in the liver.

Development of biosensors for immunoassays.

Biosensors represent a new generation of analytical instruments incorporating a biological sensing element in contact with a physical transducer and an output device. The biological element may be a whole organism, an organelle, an enzyme, an antibody, cell receptors or nucleic acids. The transducer converts the interaction between the biological component and the sample into a measurable signal which could be an electrical charge, an optical signal, a heat exchanger or some other quantifiable event. In recent years, electrochemical sensors based on continuous potentiometric or amperometric detection of enzyme reaction products have been favoured in the development of biosensors. This is because electrochemical transducers are cheap, rapid to use and portable.

Evolutionary aspects of superoxide dismutase: the copper/zinc enzyme.

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobacterium leiognathi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide dismutases shows 21 invariant residues in key positions related to maintenance of the beta-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobacterium Cu/Zn superoxide dismutase. Copper/zinc superoxide dismutase from the trematode Schistosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide, as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase, including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of beta-barrel fold and active site structure for the extracellular enzyme.

Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein.

A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.

A time-resolved fluorescence study of human copper-zinc superoxide dismutase.

The intrinsic fluorescence decay of human Cu,Zn superoxide dismutase was measured by frequency-domain techniques. The protein consists of two subunits, each containing one tryptophan and no tyrosine residues. Using a synchrotron radiation source, which allows facile selection of the excitation wavelength, the dependence of the emission decay upon excitation was studied. No significant excitation wavelength effects were found. The two tryptophans contained in the dimer, although fully equivalent and exposed to solvent, showed a fluorescence decay that cannot be described by a single lifetime. Either two lifetimes, or one Lorentzian-shaped continuous distribution of lifetimes, are needed to obtain a good fit. Under identical experimental conditions, control experiments showed that N-acetyltryptophanamide, an analogue of tryptophanyl residues in proteins, decays with a single lifetime. The heterogeneous decay of tryptophan fluorescence in superoxide dismutase is interpreted as due to the presence of static and/or dynamic conformers in the protein that decay with different lifetimes. The two models of discrete lifetimes and continuous distribution of lifetimes are discussed with reference to measurements on holo- and apo-human superoxide dismutase.

Application of the electrochemistry of cytochrome c to the measurement of superoxide radical production.

An electrochemical sensor for the measurement of the superoxide anion produced as a result of the respiratory burst of neutrophils is described. The reduction of cytochrome c by the superoxide ion was monitored using a surface-modified gold electrode. The current produced was shown to be superoxide-specific and proportional to the number of neutrophils in both purified neutrophil preparations and plasma.

Thermostable reduced nicotinamide adenine dinucleotide oxidase: application to amperometric enzyme assay.

The use in amperometric enzyme assays of a highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase), from the thermophilic organism Thermus aquaticus is described. The enzyme catalyses the oxidation of reduced nicotinamide adenine dinucleotide with concomitant two-electron reduction of dioxygen to hydrogen peroxide. In addition the enzyme used a substituted ferrocene as an alternative mediator of electron transfer. Hydrogen peroxide was detected at +650 mV vs Ag/AgCl at a platinum electrode. The current produced by oxidation of hydrogen peroxide was directly proportional to NADH concentration. The enzyme was used in solution to reoxidize enzymatically generated NADH and served as a basis for amperometric enzyme amplification systems for immunoassay as well as for the detection of substrate concentration for oxidoreductase enzymes. In the presence of alcohol dehydrogenase a rapid production of current occurred upon addition of ethanol over a clinically significant range. Thermus aquaticus NADH oxidase appears to be ideally suited for future exploitation in amperometric sensors for oxidoreductase substrates, offering a number of advantages over previously reported methods.