A novel metal-free low molecular weight superoxide dismutase mimic

2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH), the one-electron reduction product of the stable nitroxide radical, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO), is reportedly oxidized by superoxide, and its oxidation has been proposed as a method for assaying superoxide. We find that superoxide can both reduce OXANO and oxidize OXANOH. The respective rate constants, k1 and k2, were determined using two superoxide-generating systems (xanthine oxidase/xanthine as well as ionizing radiation). OXANOH oxidation and OXANO reduction are both inhibitable by superoxide dismutase, pH-dependent (4.5-9.3), and result in a steady state distribution of [OXANO] and [OXANOH], independent of their initial concentrations, i.e. the OXANO/OXANOH couple exhibits a metal-independent superoxide dismutase-like function. Thus it provides a prototype for future development of improved low molecular weight superoxide dismutase mimics which will also function in cellular hydrophobic (aprotic) compartments such as membranes.     

The hydroxylamine OXANOH and its reaction product, the nitroxide OXANO., act as complementary inhibitors of lipid peroxidation

The effects of the nitroxide 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO.) and the corresponding hydroxylamine 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) on in vitro lipid peroxidation in rat liver microsomes and reconstituted lipid vesicles were investigated, and compared with those of some commonly used spin trapping agents. OXANO. and OXANOH (10-100 microM) inhibited iron-dependent lipid peroxidation, as did the spin trapping agents (10-100 mM). OXANO. mainly inhibited the rate of peroxidation, but caused only a small delay in the time of onset. OXANOH exerted its effect by delaying the onset of peroxidation in an antioxidant fashion, and also by inhibiting the rate. Higher concentrations of both substances were required to inhibit t-butylhydroperoxide-dependent lipid peroxidation. OXANO. was found to oxidize the ferrous-ADP complex required for initiation of peroxidation, and this is probably the basis of the inhibitory effect of this compound. Since the reaction of OXANO. tends to produce OXANOH and vice versa, either one could inhibit all reactions of lipid peroxidation.     

Detection of oxygen radicals during reperfusion of intestinal cells in vitro

The nitroxide OXANO. (2-Ethyl-2,5,5-trimethyl-3-oxazolidinoxyl) which in its reduced form, OXANOH (2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine), is capable of reacting with short-lived radicals, forming a secondary stable radical, was used for ESR-detection of radical production in isolated cells. The properties of OXANO. and OXANOH in terms of stability in cellular and subcellular systems, membrane permeability and effects on cellular viability were evaluated. Ischemia and reperfusion was simulated in vitro in a preparation of cells from rat intestinal mucosa by incubation at high density (4 X 10(8) cells/ml) under an atmosphere of nitrogen for 25 min and resuspended with fresh oxygenated buffer containing 5 mM OXANOH. A significant increase in radical formation during the 15 min reperfusion period studied was obtained in cells exposed to ischemia compared to control cells incubated at normal density under an atmosphere of oxygen. The addition of 5 microM of the scavenging enzyme superoxide dismutase reduced the radical formation by 50%. The time sequence of the superoxide formation was calculated as the difference in radical production in the presence and absence of superoxide dismutase.     

Effect of CuSO4 and Cu(II)(Gly)2 on some indirect assays for superoxide dismutase activity

The effect of CuSO₄ and Cu(II)(Gly)₂ has been compared with that of superoxide dismutase on the ferricytochrome c reduction and on the nitroblue tetrazolium reduction by an enzymic or chemical flux of superoxide anion radicals as well as on o-dianisidine photooxidation. Both CuSO₄ and Cu(II)(Gly)₂ have been found to inhibit ferricytochrome c reduction as well as the aerobic and anaerobic nitroblue tetrazolium reduction with approximately equal efficiency. Unlike superoxide dismutase they proved capable of inhibiting o-dianisdine photooxidation. The effect of copper either as CuSO₄ or as Cu(II)(Cly)₂ has been established as being due to its interference with the indirect assays for superoxide dismutase activity used. The reasons for this interference have been examined and it is concluded that copper can react with a component of the indirect assay system and depending on the method used it either mimics SOD or acts contrary to the enzyme.     

Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-generating system

Alkaline dimethylsulfoxide as a superoxide anion-generating system in association with cytochrome c as a superoxide anion-indicating scavenger has been used to develop a new assay for superoxide dismutase. The assay is sensitive (one unit of enzymatic activity is provided by 110 ng of purified copper-containing superoxide dismutase) and highly specific. The nature of this system prevents the usual interferences and its simplicity allows for multiple, rapid measurements of superoxide dismutase activity in biological preparations using either normal or automated procedures.     

The Superoxide Synthases of Rose Cells : Comparison of Assays

In an effort to identify the enzymatic mechanism responsible for the synthesis of reactive oxygen species produced during the hypersensitive response, preparations of rose (Rosa damascena) cell plasma membranes, partially solubilized plasma membrane protein, and cytosol were assayed for the NADH- and NADPH-dependent synthesis of superoxide using assays for the reduction of cytochrome c (Cyt c), assays for the reduction of nitroblue tetrazolium, and assays for the chemiluminescence of N,N′-dimethyl-9,9′-biacridium dinitrate (lucigenin). Each assay ascribed the highest activity to a different preparation: the Cyt c assay to cytosol, the nitroblue tetrazolium assay to plasma membrane, and the lucigenin assay to the partially solubilized plasma membrane protein (with NADH). This suggests that no two assays measure the same set of enzymes and that none of the assays is suitable for comparisons of superoxide synthesis among different cell fractions. With the plasma membrane preparation, the presence of large amounts of superoxide-dismutase-insensitive Cyt c reductase confounded attempts to use Cyt c to measure superoxide synthesis. With the partially solubilized membrane protein, direct reduction of lucigenin probably contributed to the chemiluminescence. Superoxide synthesis detected with lucigenin should be confirmed by superoxide-dismutase-sensitive Cyt c reduction.     

Inhibitors of superoxide dismutases: A cautionary tale

An extensive search resulted in the identification of pamoic acid as an inhibitor of superoxide dismutases. Pamoic acid appeared to rapidly and reversibly inhibit all types of superoxide dismutases and did so in both the cytochrome c reduction and in the dianisidine photooxidation assays, used to measure this activity. It could nevertheless be shown that pamoic acid did not at all inhibit superoxide dismutase but rather diminished the sensitivity of the assays. The mechanism proposed to account for this effect involved oxidation of pamoate, by O₂^?, to yield a pamoate radical which can then reduce cytochrome c or oxidize pyrogallol. Pamoate thus competes with superoxide dismutase for the available O₂^?, without affecting the observable effects of that O₂^? upon cytochrome c or upon pyrogallol. It consequently makes these assays less responsive to superoxide dismutase, while appearing to be without effect in the absence of superoxide dismutase. Several of the predicted consequences of this proposal were affirmed. Other workers, interested in finding inhibitors for superoxide dismutases, are hereby forwarned of this subtle snare.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.     

Investigation of Pyrazine Formation Pathways in Glucose-Ammonia Model Systems

Model systems of d-glucose and ammonia with metal ions, oxygen, antioxidants, and sodium hydroxide were reacted at 100°C (solution temp.) for 2~18 hr to investigate pyrazine formation pathways. Pyrazines identified in these model systems were unsubstituted-, 2-methyl-, 2,5-dimethyl-, 2,6-dimethyl-, 2-ethyl-, 2,3-dimethyl-, 2-ethyl-5-methyl-, 2-ethyl-6-methyl-, 2,3,5-trimethyl-, 2-ethyl-3-methyl-, 2-vinyl-, 2-ethyl-3,5-dimethyl-, 2-ethyl-3,6-dimethyl- and ethyl vinyl-. Results show that α-amino carbonyl compounds acted as intermediates to form various pyrazines. For example, 2-methylpyrazine may be formed from the condensation of α-amino-α-hydroxy acetaldehyde and α-amino acetone following the elimination of a water molecule.

We propose that the formation of a pyrazine ring from dihydropyrazine is due to the dehydration of hydroxy dihydropyrazine rather than the dehydrogenation of dihydropyrazine. This explains the formation of an alkyl group (e.g., an ethyl group) from the sugar moiety.

We propose ten α-amino carbonyl intermediates from the alkylpyrazines which we obtained, and their formation schemes are discussed.     

The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin

A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, has been used to cross-link horse heart cytochrome c to spinach chloroplast plastocyanin. The complex was formed in yields up to 90%, and was found to have a stoichiometry of 1 mol plastocyanin per mol cytochrome c. The cytochrome c in the complex was fully reducible by ascorbate and potassium ferrocyanide, and had a redox potential only 25 mV less than that of native cytochrome c. The complex was nearly completely inactive towards succinate-cytochrome c reductase and cytochrome c oxidase, suggesting that the heme crevice region of cytochrome c was blocked. We propose that the carbodiimide promoted the formation of amide cross-links between lysine amino groups surrounding the heme crevice of cytochrome c and complementary carboxyl groups on plastocyanin. It is of interest that the high-affinity site for cytochrome c binding on bovine heart cytochrome c oxidase has recently been found to involve a sequence of subunit II with some homology to the copper-binding sequence of plastocyanin.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.     

Cytochrome c is transformed from anti- to pro-oxidant when interacting with truncated oncoprotein prothymosin α

Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones. On the other hand, in this group, it was shown that prothymosin α (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis [FEBS Lett. 467 (2000) 150]. It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin α (tPT), when meeting in cytosol, can interact with each other. In this paper, such an interaction is shown. (1) Formation of cytochrome c⋅tPT complex is demonstrated by a blot-overlay assay. (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins. The masses increase when the cytochrome c/tPT ratio increases. High concentration of KCl prevents the complex formation. (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited. (4) tPT inhibits cytochrome c oxidation by H₂O₂, catalyzed by peroxidase. Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant. A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.     

The fatty acid and sterol composition of two marine dinoflagellates

The fatty acid, sterol and chlorophyll pigment compositions of the marine dinoflagellates Gymnodinium wilczeki and Prorocentrum cordatum are reported. The fatty acids of both algae show a typical dinoflagellate distribution pattern with a predominance of C₁₈, C₂₀ and C₂₂ unsaturated components. The acid 18:5ω3 is present at high concentration in these two dinoflagellates. G. wilczeki contains a high proportion (93.4%) of 4-methyl-5α-stanols including 4,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol), dinostanol and 4,23,24-trimethyl-5α-cholest-7-en-3β-ol reported for the first time in dinoflagellates. The role of this sterol in the biosynthesis of 5α-stanols in dinoflagellates is discussed. P. cordatum contains high concentrations of a number of δ ²⁴⁽²⁸⁾-sterols with dinosterol, 24-methylcholesta-5,24(28)-dien-3β-ol, 23,24-dimethylcholesta-5,22E-dien-3β-ol, 4,24-dimethyl-5α-cholest-24(28)-en-3β-ol and a sterol identified as either 4,23,24-trimethyl- or 4-methyl-24-ethyl-5α-cholest-24(28)-en-3β-ol present as the five major components. The role of marine dinoflagellates in the input of both 4-methyl- and 4-desmethyl-5α-stanols to marine sediments is discussed.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes. Evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 × g particulate fraction of the postnuclear supernatant obtained from guinea-pig phagocytosing polymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O^?₂ generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH.2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1 : 1.3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O^?₂ generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation.4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O^?₂ formed during the oxidation of NADPH.5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH.6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH.7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible.8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase.9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O₂; an enzymatic, apparently non-univalent, O₂ reduction and a non-enzymatic chain reaction.10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O₂ is optimal at pH 7.0 with 1 mM NADPH.     

A picomolar spectrophotometric assay for superoxide dismutase

During the aerobic xanthine oxidase reaction, O₂^? is produced and accumulates to a steady state determined by a balance between the rate of production of this radical and its rate of dismutation. Addition of ferricytochrome c then results in a biphasic reduction, the very rapid phase of which reflects reaction of the accumulated O₂^?, while the slower phase corresponds to the continuing production of this radical. Superoxide dismutase suppresses the accumulation of O₂^? during the xanthine oxidase reaction and thus diminishes the burst of reduction seen upon addition of ferricytochrome c. This effect has been utilized, at pH 10.2, as the basis of an assay that permits measurement of picomolar levels of superoxide dismutase. The theory and practice of this ultrasensitive assay are described.     

Assaying for superoxide dismutase activity: Some large consequences of minor changes in conditions

Most assays for superoxide dismutase depend upon competition between the enzyme and some indicating scavenger for O₂⁻. We have investigated the effects of experimental variables on assays based upon the use of either ferricytochrome c or nitro blue tetrazolium. Our results should help investigators to avoid the numerous potential pitfalls which necessarily surround these assay methods.     

Iron-ethylenediaminetetraacetic acid (EDTA)-catalyzed superoxide dismutation revisited: An explanation of why the dismutase activity of Fe-EDTA cannot be detected in the cytochrome c/xanthine oxidase assay system

The recent assertion of J. Diguiseppi and I. Fridovich (1980, Arch. Biochem. Biophys., 203, 145–150) that Fe-EDTA does not catalyze superoxide dismutation is disputed. By directly observing superoxide generated during pulse radiolysis, we have confirmed the results of a previous study (G. J. McClune, J. A. Fee, G. A. McClusky, and J. T. Groves, 1977, J. Amer. Chem. Soc., 99, 5220–5222) which concluded that Fe-EDTA catalyzed superoxide dismutation. We also demonstrate that the reaction of Fe(II)-EDTA, formed during catalyzed superoxide dismutation, with cytochrome c, the probe molecule in the cytochrome c/xanthine oxidase/xanthine assay system for superoxide dismutase activity, is sufficiently rapid (H. L. Hodges, R. A. Holwerda, and H. B. Gray, 1974, J. Amer. Chem. Soc., 96, 3132–3137) to obscure the weak catalysis of superoxide dismutation by Fe-EDTA.     

Different reactivity of carboxylic groups of cytochrome c oxidase polypeptides from pig liver and heart

Cytochrome c oxidase isolated from pig liver and heart was incubated with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide and [¹⁴C]glycine ethyl ester in the presence and absence of cytochrome c. Labelling of individual subunits was determined after separation of the enzyme complexes into 13 polypeptides by SDS-gel electrophoresis. Polypeptide II and additional but different polypeptides were labelled in the liver and in the heart enzyme. Labelling of polypeptide II and of some other polypeptides could be partially or completely suppressed by cytochrome c. From the data two conclusions can be drawn: In addition to polypeptide II, other polypeptides take part in the binding of cytochrome c to cytochrome c oxidase; the binding domain for cytochrome c is different in pig liver and heart cytochrome c oxidase.Cytochrome c oxidase isozymeCytochrome c binding domain1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideTissue specificity     

Chemical cross-linking of ferredoxin to spinach thylakoids: Evidence for two independent binding sites of ferredoxin to the membrane

Ferredoxin has been chemically cross-linked to thylakoids by using N-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The membranes thus treated became able to photoreduce cytochrome c and to catalyze the NADPH-cytochrome c reductase reaction without adding exogenous ferredoxin. Preincubation of thylakoids with an antibody against ferredoxin-NADP⁺ reductase before carbodiimide treatment or removal of the reductase by mild trypsin treatment after the cross-linking reaction did not alter the cytochrome c photoreduction activity of the treated membranes. Two independent binding sites of ferredoxin to thylakoids are thus inferred: one site is shown to be the membrane-bound reductase, the second is suggested to be at the level of the photosystem I complex.     

Mechanistic insights into the superoxide–cytochrome c reaction by lysine surface scanning

This study summarizes results which have been obtained by a mutational study of human cytochrome c. The protein can be used as a recognition element in analytical assays and biosensors for superoxide radicals since ferricytochrome c reacts with superoxide to form ferrocytochrome c and oxygen. Here lysine mutagenesis of the distal surface (i.e., of exposed residues around the Met80 axial ligand) of human cytochrome c was pursued to evaluate the effect of the surface charges on the reaction rate with the superoxide anion radical and on the redox properties of the heme protein. The latter feature is particularly relevant when the protein is immobilized on a negatively charged self-assembled monolayer on an electrode to be used as a biosensor. The observed effects of the mutations are rationalized through structural investigations based on solution NMR spectroscopy and computational analysis of the surface electrostatics. The results suggest the presence of a specific path that guides superoxide toward an efficient reaction site. Localized positive charges at the rim of the entry channel are effective in increasing the reaction rate, whereas diffused positive charges or charges far from this area are not effective or are even detrimental, resulting in a misguided approach of the anion to the protein surface.