A sensitive assay for superoxide dismutase based on the autoxidation of 6-hydroxydopamine

A simple, sensitive spectrophotometric assay system for superoxide dismutase (SOD) has been developed. This assay is based on the inhibitory effects of SOD on the initial rate of 6-hydroxydopamine autoxidation. The inhibition of 6-hydroxydopamine autoxidation was virtually linear to an SOD concentration of approximately 100 ng of SOD/ml (about a 50% inhibition at 100 ng/ml; there was a greater inhibition at higher SOD concentrations). With this assay system it was determined that SOD levels in rat brain, liver, and spinal cord were 84, 660, and 56 μg of SOD/g of tissue, respectively. These results agree very well with results obtained by other assays.     

Effect of superoxide dismutase on the autoxidation of various hydroquinones--a possible role of superoxide dismutase as a superoxide:semiquinone oxidoreductase

The autoxidation of DT-diaphorase-reduced 1,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2-OH-p-benzoquinone is efficiently prevented by superoxide dismutase. This effect was assessed in terms of an inhibition of NADPH oxidation (over the amount required to reduce the available quinone), O2 consumption, and H2O2 formation. Superoxide dismutase also affects the distribution of molecular products -hydroquinone/quinone-involved in autoxidation, by favoring the accumulation of the reduced form of the above quinones. In contrast, the rate of autoxidation of DT-diaphorase-reduced 1,2-naphthoquinone is enhanced by superoxide dismutase, as shown by increased rates of NADPH oxidation, O2 consumption, and H2O2 formation and by an enhanced accumulation of the oxidized product, 1,2-naphthoquinone. These findings suggest that superoxide dismutase can either prevent or enhance hydroquinone autoxidation. The former process would imply a possible new activity displayed by superoxide dismutase involving the reduction of a semiquinone by O2-.. This activity is probably restricted to the redox properties of the semiquinones under study, as indicated by the failure of superoxide dismutase to prevent autoxidation of 1,2-naphthohydroquinone.     

Modeling the influence of superoxide dismutase on superoxide and nitric oxide interactions,including reversible inhibition of oxygen consumption

A mathematical mass transport model was constructed in cylindrical geometry to follow coupled biochemical reactions and diffusion of oxygen, nitric oxide, superoxide, peroxynitrite, hydrogen peroxide, nitrite, and nitrate around a blood vessel. Computer simulations were performed for a 50 microm internal diameter arteriole to characterize mass transport in five concentric regions (blood, plasma layer, endothelium, vascular wall, perivascular tissue). Steady state gradients in nitric oxide, oxygen partial pressure, superoxide, and peroxynitrite, and associated production of hydrogen peroxide, nitrite, and nitrate were predicted for varying superoxide production rates, superoxide dismutase concentrations, and other physiological conditions. The model quantifies how competition between superoxide scavenging by nitric oxide and superoxide dismutase catalyzed removal varies spatially. Reversible inhibition of oxygen consumption by nitric oxide, which causes increased tissue oxygenation at deeper locations, was also included in the model. The mass transport model provides insight into complex interactions between reactive oxygen and nitrogen species in blood and tissue, and provides an objective way to evaluate the relative influence of different biochemical pathways on these interactions.     

Effect of superoxide dismutase on the autoxidation of substituted hydro- and semi-naphthoquinones

The effect of superoxide dismutase on the autoxidation of hydro- and semi-1,4-naphthoquinones with different substitution pattern and covering a one-electron reduction potential range from -95 to -415 mV was examined. The naphthoquinone derivatives were reduced via one or two electrons by purified NADPH-cytochrome P-450 reductase or DT-diaphorase, respectively. Superoxide dismutase did not alter or slightly enhance the initial rates of enzymic reduction, whereas it affected in a different manner the following autoxidation of the semi- and hydroquinones formed. Autoxidation was assessed as NADPH oxidation in excess to the amounts required to reduce the quinone present, H2O2 formation, and the redox state of the quinones. Superoxide dismutase enhanced 2--8-fold the autoxidation of 1,4-naphthosemiquinones, following the reduction of the oxidized counterpart by NADPH-cytochrome P-450 reductase, except for the glutathionyl-substituted naphthosemiquinones, whose autoxidation was not affected by superoxide dismutase. Superoxide dismutase exerted two distinct effects on the autoxidation of naphthohydroquinones formed during DT-diaphorase catalysis: on the one hand, it enhanced slightly the autoxidation of 1,4-naphthohydroquinones with a hydroxyl substituent in the benzene ring: 5-hydroxy-1,4-naphthoquinone and the corresponding derivatives with methyl- and/or glutathionyl substituents at C2 and C3, respectively. On the other hand, superoxide dismutase inhibited the autoxidation of naphthohydroquinones that were either unsubstituted or with glutathionyl-, methyl-, methoxyl-, hydroxyl substituents (the latter in the quinoid ring). The inhibition of hydroquinone autoxidation was reflected as a decrease of NADPH oxidation, suppression of H2O2 production, and accumulation of the reduced form of the quinone. The enhancement of autoxidation of 1,4-naphthosemiquinones by superoxide dismutase has been previously rationalized in terms of the rapid removal of O2-. by the enzyme from the equilibrium of the autoxidation reaction (Q2-. + O2----Q + O2-.), thus displacing it towards the right. The superoxide dismutase-dependent inhibition of H2O2 formation as well as NADPH oxidation during the autoxidation of naphthohydroquinones--except those with a hydroxyl substituent in the benzene ring--seems to apply to those organic substrates which can break down with simultaneous formation of a semiquinone and O2-.. Inhibition of hydroquinone autoxidation by superoxide dismutase can be interpreted in terms of suppression by the enzyme of O2-.- dependent chain reactions or a direct catalytic interaction with the enzyme that might involve reduction of the semiquinone at expense of O2(-.).(ABSTRACT TRUNCATED AT 400 WORDS)     

The stimulation of 6-hydroxydopamine autoxidation by bivalent copper: Potential importance in the neurotoxic process

Copper sulfate (Cu²⁺, 10^?6M to 10^?5M) stimulated the rate of autoxidation of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) as judged both by absorbancy measurements at 490 nm and oxygen consumption. This stimulation of 6-OHDA autoxidation by Cu²⁺ was prevented by the copper chelating agent EDTA. In other experiments, Cu²⁺ stimulated the rates of autoxidation of dopamine and norepinephrine as well as the neurotoxic agents 6-aminodopamine, 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine. It is suggested that intraneuronal levels of copper, by virtue of controlling the rates of autoxidation reactions, might be important in vivo.     

Competitive inhibition of Cu, Zn superoxide dismutase by monovalent anions.

Polarographic measurements showed that N₃^? and halides $\text{in}$ hibit the activity of bovine Cu, Zn superoxide dismutase in a competitive fashion, as previously demonstrated for CN^? and OH^?. All anions increase the spin-lattice nuclear magnetic relaxation time (T₁) of aqueous solutions of the enzyme as well, but the stability constants measured from T₁ data are lower than those calculated from activity data. The results suggest that substrate and anionic inhibitors bind during the catalytic action at the water coordination position of the enzyme copper, and that these inhibitors may have a greater affinity for the cuprous form of the enzyme which is generated in the catalytic cycle.     

Lactobacillus sanfranciscensis CB1: manganese, oxygen, superoxide dismutase and metabolism

The latency phase, growth rate, cell yield and end-products of Lactobacillus sanfranciscensis CB1 were affected by oxygen and the supply of 225 μM Mn²⁺. Mn²⁺ was especially related to the highest substrate consumption. Aerobiosis and Mn²⁺ supply were responsible for the highest superoxide dismutase activity. An auto-inhibitory accumulation of H₂O₂ meant that the survival of air-grown cells supplied with Mn²⁺ rapidly decreased during the stationary phase. As shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Mn²⁺ supply influenced protein expression. As shown by non-denaturating zymograms, Lb. sanfranciscensis CB1 expressed an approximately 12.5-kDa superoxide dismutase, which is probably Mn-dependent. The enzyme was insensitive to H₂O₂ treatment, was not induced by the presence of paraquat under aerobic conditions and was relatively stable at pH 4.0. Sourdoughs that contained high levels of oxygen enhanced cell growth, acidification and acetic acid production by Lb. sanfranciscensis CB1. Received: 24 July 1998 / Received last revision: 11 November 1998 / Accepted: 13 November 1998     

Intermediates in the aerobic autoxidation of 6-hydroxydopamine: relative importance under different reaction conditions

Autoxidation of 6-hydroxydopamine (6-OHDA) proceeds through a balanced network of: transition metal ions, superoxide, hydrogen peroxide, hydroxyl radicals, and other species. The contribution of each to the reaction mechanism varies dramatically depending upon which scavengers are present. The contribution of each propagating intermediate increases when the involvement of others is diminished. Thus, superoxide (which is relatively unimportant when metal ions can participate) dominates the reaction when transition metal ions are bound (especially at higher pH), and it becomes essential in the simultaneous presence of catalase plus chelators. Transition metal ions participate more if superoxide is excluded; hydrogen peroxide becomes more important if both .O2- and metal ions are excluded; and hydroxyl radicals contribute more to the reaction mechanism if both H2O2 and .O2- are excluded. Superoxide dismutase inhibited strongly, by two distinct mechanisms: a high affinity mechanism (less than 13% inhibition) at catalytically effective concentrations, and a low affinity mechanism (almost complete inhibition at the highest concentrations) which depends upon both metal binding and catalytic actions. In the presence of DETAPAC catalytic concentrations of superoxide dismutase inhibited by over 98%. Conversely, metal chelating agents inhibited strongly in the presence of superoxide dismutase. When present alone they stimulated (like EDTA), inhibited (like desferrioxamine), or had little effect (like DETAPAC). Catalase which stimulated slightly but consistently (less than 5%) when added alone, inhibited 100% in the presence of superoxide dismutase + DETAPAC. However, in the absence of DETAPAC, catalase decreased inhibition by superoxide dismutase, yielding a 100% increase in reaction rate. Hydroxyl scavengers (formate, mannitol or glucose) alone produced little or no (less than 10%) inhibition, but inhibited by 30% in the presence of catalase + superoxide dismutase. Paradoxically, they stimulated the reaction in the presence of catalase + superoxide dismutase + DETAPAC.     

In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate.

Superoxide dismutase was assayed by a method which takes advantage of the inhibitory action of superoxide dismutase (or tissues which contain superoxide dismutase) on the rate of autooxidation of 6-hydroxydopamine. Incubation of pure superoxide dismutase of homogenates of brain or liver with 10(-3) M diethyldithiocarbamate for 1.5 hours resulted in total loss of superoxide dismutase activity. Inhibition of superoxide dismutase was not reversed by dialysis, but after dialysis, enzymatic activity was restored with CuSO4. When 1.5 g of diethyldithiocarbamate/kg were injected into mice, the superoxide dismutase activity at 3 hours was decreased by 86%, 71%, and 48%, respectively, in whole blood, liver, and brain. A dose of 0.5 g of diethyldithiocarbamate/kg lowered the superoxide dismutase activity by 42% in liver at 3 hours. A study of the time course for inhibiton of superoxide dismutase in liver after 1.5 g of diethyldithiocarbamate/kg, showed a maximum decrease (81%) within 1 hour, with a slow return to 64% of normal by 24 hours. Inhibition of superoxide dismutase in vivo and in vitro was confirmed with other assay systems based on the autooxidation of pyrogallol or epinephrine or on reduction of cytochrome c or intro blue tetrazolium. Treatment of animals with diethyldithiocarbamate may provide a useful experimental model to study the role of superoxide dismutase in various tissues.     

Phosphate inhibition of the copper- and zinc-containing superoxide dismutase: a reexamination

Phosphate was reported to be an inhibitor of copper- and zinc-containing superoxide dismutase (SOD) [de Freitas, D.M., & Valentine, J.S. (1984) Biochemistry 23, 2079-2082]. Thus SOD activity, in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), was decreased by approximately 50% when the assay was made 10 mM in phosphate, and the ionic strength was adjusted with sodium fluoride. The inhibitory effect of phosphate was attributed to the neutralization of the positive charge on the guanidino residue of Arg-141. We have reexamined the effects of phosphate inhibition of SOD and found that the enzyme has identical activity in phosphate or HEPES buffer when the ionic strength is adjusted with NaBr. The putative inhibitory effect of phosphate appears to have been due to fluoride inhibition of the superoxide generating system of xanthine/xanthine oxidase. We have confirmed this result by using a photochemical generation of O2- in addition to the enzymatic generation of O2-. Chemical modification of the lysine residues to homoarginines does not affect the activity of the enzyme and does not impart a phosphate sensitivity. Chemical modification with phenylglyoxal caused approximately 80% inactivation of the native enzyme and 90% inactivation of the O-methylisourea-modified enzyme. Our results suggest that phosphate does not inhibit the copper- and zinc-containing superoxide dismutase (Cu,Zn-SOD) beyond the expectations of its effect on ionic strength.     

Mycoplasma pneumoniae: A prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase

Superoxide dismutase and catalase were not detected in $\text{M}$. $\text{pneumoniae}$ and several other species of $\text{Mycoplasma}$ some of which consume oxygen and secrete H₂O₂. $\text{M}$. $\text{pneumoniae}$ in suspension formed O₂^? in the presence of NADH and flavins and extracts of $\text{M}$. $\text{pneumoniae}$ formed O₂^? in the presence of either NADH or NADPH. The lack of superoxide dismutase in $\text{M}$. $\text{pneumoniae}$ could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O₂ synthesize superoxide dismutase.     

Manganese, superoxide dismutase, and oxygen tolerance in some lactic acid bacteria

A previous study of the aerotolerant bacterium Lactobacillus plantarum, which lacks superoxide dismutase (SOD), demonstrated that it possesses a novel substitute for this defensive enzyme. Thus, L. plantarum contains 20 to 25 mM Mn(II),m in a dialyzable form, which is able to scavenge O2- apparently as effectively as do the micromolar levels of SOD present in most other organisms. This report describes a survey of the lactic acid bacteria. The substitution of millimolar levels of Mn(II) for micromolar levels of SOD is a common occurrence in this group of microorganisms, which contained either SOD or high levels of Mn(II), but not both. Two strains were found which had neither high levels of Mn(II) nor SOD, and they were, as was expected, very oxygen intolerant. Lactic acid bacteria containing SOD grew better aerobically than anaerobically, whereas the organisms containing Mn(II) in place of SOD showed aerobic growth which was best, at best, equal to anaerobic growth. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) increases the rate of O2- production in these organisms. Lactobacillus strains containing high intracellular Mn(II) were more resistant to the oxygen-dependent toxicity of plumbagin than were strains containing lower levels of Mn(II). The results support the conclusion that a high internal level of Mn(II) provides these organisms with an important defence against endogenous O2-.     

Apparent oxygen-dependent inhibition by superoxide dismutase of the quinoprotein methanol dehydrogenase.

Methanol dehydrogenase activity, when assayed with phenazine ethosulfate (PES) as an electron acceptor, was inhibited by superoxide dismutase (SOD) and by Mn2+ only under aerobic conditions. Catalase, formate, and other divalent cations did not inhibit the enzyme. The enzyme also exhibited significantly higher levels of activity when assayed with PES under anaerobic conditions relative to aerobic conditions. The oxygen- and superoxide-dependent effects on methanol dehydrogenase were not observed when either Wurster's Blue or cytochrome c-55li was used as an electron acceptor. Another quinoprotein, methylamine dehydrogenase, which possesses tryptophan tryptophylquinone (TTQ) rather than pyrroloquinoline quinone (PQQ) as a prosthetic group, was not inhibited by SOD or Mn2+ when assayed with PES as an electron acceptor. Spectroscopic analysis of methanol dehydrogenase provided no evidence for any oxygen- or superoxide-dependent changes in the redox state of the enzyme-bound PQQ cofactor of methanol dehydrogenase. To explain these data, a model is presented in which this cofactor reacts reversibly with oxygen and superoxide, and in which oxygen is able to compete with PES as an electron acceptor for the reduced species.     

Kinetic stability of Cu/Zn superoxide dismutase is dependent on its metal ligands: implications for ALS

Over 100 mutants of the enzyme Cu/Zn superoxide dismutase (SOD) have been implicated in the neurodegenerative disease familial amyotrophic lateral sclerosis (FALS). Growing evidence suggests that the aggregation of SOD mutants may play a causative role in FALS and that aberrant copper chemistry, decreased thermodynamic stability, and decreased affinity for metals may contribute independently or synergistically to this process. Since the loss of the copper and zinc ions significantly decreases the thermodynamic stability of SOD, it is expected that this would also decrease its kinetic stability, thereby facilitating partial or global unfolding transitions that may lead to misfolding and aggregation. Here we used wild-type (WT) SOD and five FALS-related mutants (G37R, H46R, G85R, D90A, and L144F) to show that the metals contribute significantly to the kinetic stability of the protein, with demetalated (apo) SOD showing acid-induced unfolding rates about 60-fold greater than the metalated (holo) protein. However, the unfolding rates of SOD WT and mutants were similar to each other in both the holo and apo states, indicating that regardless of the effect of mutation on thermodynamic stability, the kinetic barrier toward SOD unfolding is dependent on the presence of metals. Thus, these results suggest that pathogenic SOD mutations that do not significantly alter the stability of the protein may still lead to SOD aggregation by compromising its ability to bind or retain its metals and thereby decrease its kinetic stability. Furthermore, the mutant-like decrease in the kinetic stability of apo WT SOD raises the possibility that the loss of metals in WT SOD may be involved in nonfamilial forms of ALS.     

Neuronal transfer of the human Cu/Zn superoxide dismutase gene increases the resistance of dopaminergic neurons to 6-hydroxydopamine

Several mechanisms are thought to be involved in the progressive decline in neurons of the substantia nigra pars compacta (SNpc) that leads to Parkinson's disease (PD). Neurotoxin 6-hydroxydopamine (6-OHDA), which induces parkinsonian symptoms in experimental animals, is thought to be formed endogenously in patients with PD through dopamine (DA) oxidation and may cause dopaminergic cell death via a free radical mechanism. We therefore investigated protection against 6-OHDA by inhibiting oxidative stress using a gene transfer strategy. We overexpressed the antioxidative Cu/Zn-superoxide dismutase (SOD1) enzyme in primary culture dopaminergic cells by infection with an adenovirus carrying the human SOD1 gene (Ad-hSOD1). Survival of the dopaminergic cells exposed to 6-OHDA was 50% higher among the SOD1-producing cells than the cells infected with control adenoviruses. In contrast, no significant increased survival of (6-OHDA)-treated dopaminergic cells was observed when they were infected with an adenovirus expressing the H(2) O(2) -scavenging glutathione peroxidase (GPx) enzyme. These results underline the major contribution of superoxide in the dopaminergic cell death process induced by 6-OHDA in primary cultures. Overall, this study demonstrates that the survival of the dopaminergic neurons can be highly increased by the adenoviral gene transfer of SOD1. An antioxidant gene transfer strategy using viral vectors expressing SOD1 is therefore potentially beneficial for protecting dopaminergic neurons in PD.