Superoxide dismutase and peroxidase: a positive activity stain applicable to polyacrylamide gel electropherograms.

The oxidation of dianisidine, photosensitized by riboflavin, is accelerated by superoxide dismutase. Polyacrylamide gel electropherograms soaked in riboflavin plus dianisidine and subsequently illuminated develop stable brown bands at positions bearing superoxide dismutase activity. This constitutes a new, convenient, and advantageous activity stain for this class of enzymes. Peroxidases are also stained by this procedure due to the photochemical production of H₂O₂. This does not constitute an interference with the specificity of the stain, since peroxidase bands develop more slowly than superoxide dismutase bands and can be further identified through the use of inhibitors or of independent staining for peroxidase. The new, positive activity stain for superoxide dismutases can be applied to crude extracts of cells.     

An ultrasensitive colorimetric assay for manganese

An ultrasensitive colorimetric assay for manganese is described. It is based upon the catalysis, by Mn(II), of the photochemical oxidation of o-dianisidine, sensitized by riboflavin. Catalase increases the Mn(II)-catalyzed rate of photosensitized oxidation of dianisidine to the bisazobiphenyl, while superoxide dismutase inhibits the rate. The mechanism appears to involve oxidation of Mn(II) by O2-, followed by oxidation of dianisidine by MnO2+ in equilibrium Mn(III). Cu(II) interferes, but Zn(II), Fe(II), Fe(III), Co(II), and Ni(II) do not. Chelating agents and thiol reductants also interfere. Interference by Cu(II) can be overcome by the addition of cyanide, while interference by organic compounds can be surmounted by wet ashing. This assay provides a linear response to Mn(II) over the range 10-2500 nM. The limit of detection was 5 nM Mn(II).     

Retardation of photo-induced changes in Photosystem I submembrane particles by glycinebetaine and sucrose

The protective role of co-solutes (glycinebetaine and sucrose) against photodamage in isolated Photosystem (PS) I submembrane particles illuminated (2000 μE m⁻² s⁻¹) for various time periods at 4 °C was studied. The photochemical activity of PS I in terms of electron transport measured as oxygen uptake and P700 photooxidation was significantly protected. A photoinduced enhancement of oxygen uptake observed during the first hours of strong light illumination attributed to denaturation or dissociation of membrane-bound superoxide dismutase [Rajagopal et al. (2003) Photochem. Photobiol 77: 284–291] was also retarded by glycinebetaine and sucrose. Chlorophyll photobleaching resulting in a decrease of absorbance and a blue-shift of the absorbance maximum in the red was greatly delayed in the presence of co-solutes. This phenomenon was also observed in the chlorophyll-protein (CP) complexes of PS I particles exposed to strong illumination separated on non-denaturing poly-acrylamide gels. In this case, a decrease in the absorbance of the CP1b band coinciding with an increase of CP1a during the course of illumination and ascribed to oxidative cross-linking (Rajagopal et al. 2003) was also retarded. Our results, thus, clearly demonstrated for the first time that co-solutes could minimize the alteration of photochemical activity and chlorophyll-protein complexes against photodamage of PS I submembranes particles. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assessment of the genotoxic potential of riboflavin and lumiflavin. B. Effect of light.

On exposure to visible light, riboflavin and lumiflavin produced reactive oxygen species such as singlet oxygen and superoxide radicals. The reaction was found to be time- and concentration-dependent. Both riboflavin and lumiflavin, upon illumination, showed mutagenic response in the umu test as well as in the Ames/Salmonella assay with Salmonella typhimurium TA102. The mutagenic response was partially abolished by superoxide dismutase while sodium azide did not have any effect. No mutagenicity was observed if the compounds were not illuminated. The results suggested the involvement of superoxide radicals in light-induced mutagenicity of riboflavin as well as lumiflavin.     

Leukotriene B4 and its action with a free-radical-generating system

The concentration of Leukotriene B₄ (LTB₄) demonstrated in early inflammation has been shown to induce leukocyte aggregation, chemotaxis and degranulation of polymorphonuclear leukocytes (PMN) $\text{in vitro}$. N-f-Met. Leu-Phe, a potent chemotactic factor, has been shown to activate neutrophils to produce chemiluminescence and produce superoxide radicals. The characteristics of the LTB₄-induced degranulation of rabbit neutrophils are strikingly similar to those of the chemotactic factors. Thiols, and in partiicular glutathione, have been shown to have a marked inhibitory effect in clinical assays of superoxide dismutase (SOD) activity, using reactions which are supposedly specific for the superoxide ion. SOD is most frequently assessed by coupling a generator of O₂^? with an indicating scavenger for the radical. The enzyme then competes with the scavenger for the available O₂^? and inhibits the processes being observed, thus, the inhibition serves as a basis for estimation of SOD activity. A method proposed by Misra and Fridovich for the estimation of SOD activity is based on the photo-oxidation of dianisidine sensitised by riboflavin.This assay can be used to classify compounds as either SOD-like or glutathione-like. With a small quantity if LTB₄ and LTD₄, we obtained preliminary results for their effect on the assay (Table 1). They appear to be glutathione-like, i.e., reactive with the free-radical-generating system in preference to a specific reaction with O₂^? and are only slightly less effective than glutathione.Although our results are preliminary it is clear that the leukotrienes are effective as radical scavengers in this reaction. Further studies with two prostaglandis (products of the cycloxygenase pathway) will also be presented.     

Kinetic study on the photostability of riboflavin in the presence of barbituric acid

Abstract

The photochemical fate of riboflavin (vitamin B₂) in the presence of barbituric acid was examined employing polarographic detection of dissolved oxygen and steady-state and time-resolved spectroscopy. Under visible light, riboflavin reacts with barbituric acid – the latter being transparent to this type of photo-irradiation – via radicals and reactive oxygen species, such as singlet molecular oxygen [O₂(¹Δ_g)] and superoxide radical anion, which are generated from the excited triplet state of the vitamin. As a result, both the vitamin and barbituric acid are photodegraded. Kinetic and mechanistic studies on the photoreactions of riboflavin in the presence of barbituric acid indicate the excellent quenching ability of the latter towards O₂(¹Δ_g).     

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.     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance     

Superoxide dismutases: I. Occurrence in higher plants

Shoots, roots, and seeds of corn (Zea mays L., cv. Michigan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were analyzed for their superoxide dismutase content using a photochemical assay system consisting of methionine, riboflavin, and p-nitro blue tetrazolium. The enzyme is present in the shoots, roots, and seeds of the three species. On a dry weight basis, shoots contain more enzyme than roots. In seeds, the enzyme is present in both the embryo and the storage tissue. Electrophoresis indicated a total of 10 distinct forms of the enzyme. Corn contained seven of these forms and oats three. Peas contained one of the corn and two of the oat enzymes. Nine of the enzyme activities were eliminated with cyanide treatment suggesting that they may be cupro-zinc enzymes, whereas one was cyanide-resistant and may be a manganese enzyme. Some of the leaf superoxide dismutases were found primarily in mitochondria or chloroplasts. Peroxidases at high concentrations interfere with the assay. In test tube assays of crude extracts from seedlings, the interference was negligible. On gels, however, peroxidases may account for two of the 10 superoxide dismutase forms.     

Generation of superoxides during the interaction of melanins with oxygen

The rate of nitroblue tetrazolium (NBT) reduction by dihydroxyphenylalanine-melanin, pheomelanin and retinal pigment epithelium melanosomes under aerobic conditions (pH 7.4) is low both in the dark and upon illumination, but increases drastically in the presence of cetyltrimethylammonium bromide (CTAB). Under these conditions, the light insignificantly stimulates NBT reduction (1.3-fold). The reaction is effectively inhibited by superoxide dismutase. This suggests that superoxide anions (O2-. are formed as intermediate reaction products in the course of NBT reduction by melanins. At alkaline values of pH (greater than or equal to 9.0), the O2-.-dependent reduction of NBT can also take place in the absence of CTAB. In contrast with oxidation of photoreduced riboflavin, the melanin oxidation by O2 cannot induce lipid peroxidation. It is concluded that O2-. generation via melanin oxidation of melanosomes occurs only under non-physiological conditions and can hardly take place in vivo.     

Protective effect of active oxygen scavengers on protein degradation and photochemical function in photosystem I submembrane fractions during light stress

The protective role of reactive oxygen scavengers against photodamage was studied in isolated photosystem (PS) I submembrane fractions illuminated (2000 microE x m(-2) x s(-1)) for various periods at 4 degrees C. The photochemical activity of the submembrane fractions measured as P700 photooxidation was significantly protected in the presence of histidine or n-propyl gallate. Chlorophyll photobleaching resulting in a decrease of absorbance and fluorescence, and a blue-shift of both absorbance and fluorescence maximum in the red region, was also greatly delayed in the presence of these scavengers. Western blot analysis revealed the light harvesting antenna complexes of PSI, Lhca2 and Lhca1, were more susceptible to strong light when compared to Lhca3 and Lhca4. The reaction-center proteins PsaB, PsaC, and PsaE were most sensitive to strong illumination while other polypeptides were less affected. Addition of histidine or n-propyl gallate lead to significant protection of reaction-center proteins as well as Lhca against strong illumination. Circular dichroism (CD) spectra revealed that the alpha-helix content decreased with increasing period of light exposure, whereas beta-strands, turns, and unordered structure increased. This unfolding was prevented with the addition of histidine or n-propyl gallate even after 10 h of strong illumination. Catalase or superoxide dismutase could not minimize the alteration of PSI photochemical activity and structure due to photodamage. The specific action of histidine and n-propyl gallate indicates that 1O2 was the main form of reactive oxygen species responsible for strong light-induced damage in PSI submembrane fractions.     

A rapid whole-cell assay for superoxide dismutase

A procedure is described for the intact-cell assay of superoxide dismutase(s). The technique involves the use of toluene which renders the cells permeable to the necessary components of a photochemical assay for superoxide dismutase. Whole-cell superoxide dismutase activities from a number of procaryotic and eucaryotic microorganisms compare with cell-free activities and with activities reported in the literature. Using this procedure, changing levels of superoxide dismutase are readily monitored under conditions known to modulate superoxide dismutase activity assayed in vitro. In whole cells of Escherichia coli, exogenous methyl viologen causes a marked increase in superoxide dismutase activity, whereas in the cyanobacterium, Microcystis aeruginosa, such treatment leads to a marked, light-dependent loss of whole-cell superoxide dismutase activity.     

Induction of aryl hydrocarbon hydroxylase by a light-driven superoxide generating system in liver cell culture

Aryl Hydrocarbon Hydroxylase activity can be increased in rat liver cell cultures by the generation of the superoxide ion (O₂^?) in the growth medium. This can be achieved by mild illumination of the cells in the presence of riboflavin and methionine. The increased activity that results can be prevented by Cycloheximide and appears to be a typical induction. It is suggested that superoxide generation within cells may be a common factor linking different microsomal enzyme inducers.     

Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the non-flavinogenic yeast Saccharomyces cerevisiae.     

The oxidation of phenylhydrazine: superoxide and mechanism.

The oxidation of phenylhydrazine in buffered aqueous solutions is a complex process involving several intermediates. It can be initiated by metal cations, such as Cu2+; in which case EDTA acts as an inhibitor. It can also be intiated by oxyhemoglobin; in which case chelating agents do not interfere. Superoxide radical is both a product of this reaction and a chain propagator. The formation of O2- could be demonstrated in terms of a reduction of nitroblue tetrazolium, which was prevented by superoxide dismutase. The importance of O2- in carrying the reaction chains was shown by the inhibition of phenylhydrazine oxidation by superoxide dismutase. Hydrogen peroxide accumulated during the reaction and could be detected with catalase. The progress of this oxidation could be monitored in terms of oxygen consumption and by following increases in absorbance at 280 or 320 nm. The oxidation was markedly autocatalytic and superoxide dismutase had the effect of extending the lag period. The absorbance at 280 nm was due to an intermediate which first accumulated and was then consumed. This intermediate appears to be benzendiazonium ion. The absorbance at 320 nm was due to a stable product, which was not identified. The time course of oxygen consumption paralleled the increase in absorbance at 320 nm and lagged behind the changes at 280 nm. Exogenous benzenediazonium ion accelerated the oxidation of phenylhydrazine and eliminated the lag phase. Benzenediazonium ion must therefore react with phenylhydrazine to produce a very reactive intermediate, possibly phenyldiazene. A mechanism was proposed which is consistent with the data. The intermediates and products of the oxidation of phenylhydrazine include superoxide radical, hydrogen peroxide, phenylhydrazyl radical, phenyldiazene, and benzenediazonium ion. This is a minimal list: others remain to be detected and identified. It appears likely that the diverse biological effects of phenylhydrazine are largely due to the reactivities of these intermediates and products.     

Chloroplast manganese and superoxide.

Particles prepared from spinach chloroplast membranes with Triton X-100 inhibited the superoxide-mediated reduction of nitro-blue tetrazolium by riboflavin. This superoxide dismutase-like activity was of two kinds, one inactivated by heating and inhibited by H₂O₂ and the other insensitive to both of these treatments; both activities were destroyed by washing with concentrated Tris buffer or with EDTA. Attempts at reconstitution with transition metal ions suggested that two different forms of bound manganese may be responsible and it is proposed that the inhibition by H₂O₂ is indicative of three different oxidation states of particle-bound manganese. The possibility that the photosynthetic water-splitting system and superoxide dismutase have evolved from a single precursor is discussed.     

In vitro photochemical cataract in mice lacking copper-zinc superoxide dismutase

We here evaluate cataract formation in mice lacking the cytosolic copper-zinc superoxide dismutase (CuZn-SOD) in an in vitro model using irradiation with visible light and riboflavin as a photosensitizing agent. Isolated, cultured lenses from wild-type and CuZn-SOD-null mice were irradiated for 1.5 h by a daylight fluorescent light after preincubation with 10 microM riboflavin for 24 h. Cataract formation was evaluated daily with digital image analysis and ocular staging, and after 5 d 86Rb uptake and water contents of the lenses were determined. Basal superoxide concentrations in freshly isolated lenses from wild-type and CuZn-SOD-null mice were determined with lucigenin-derived chemiluminescense, and enzymatic activities of all three SOD isoenzymes in the murine lens were determined with a direct spectrophotometric method. The cytosolic CuZn-SOD accounts for 90% of the total SOD activity of the murine lens. CuZn-SOD-null lenses showed a doubled basal superoxide concentration, and were more prone to develop photochemical cataract in the present model with more opacification, more hydration, and less 86Rb uptake than lenses from wild-type mice. We conclude that CuZn-SOD is an important superoxide scavenger in the lens, and that it may have a protective role against cataract formation.     

Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin.

The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte.     

The quenching effect of blue light on halorhodopsin

A mutant of Halobacterium halobium which contains halorhodopsin was isolated from strain S₉. An absorbance change at 380 nm caused by steady orange light illumination (λ ?530 nm) was observed. This change depended upon the intensity of the actinic light. The bleached envelope vesicles and vesicles derived from nicotine-grown cells showed a small or no absorbance change at 380 nm, suggesting that the change stemmed from the photochemical intermediate of halorhodopsin (referred to as P-380). When blue light was superimposed on orange background illumination, the membrane potential (Δψ) of the envelope vesicles decreased. Δψ was determined from the tetraphenylphosphonium cation (TPP⁺) distribution by means of a TPP⁺ electrode. When blue light intensity was increased, both Δψ and the amount of P-380 were decreased. An equation was derived which showed that Δψ is proportional to the concentration of P-380 formed by illumination under the assumption that the ionic composition is not significantly changed upon illumination. This equation was checked experimentally from the following three points: The blue light effect, the relationship between Δψ and light intensity, and the effect of gramicidin. The data obtained accorded well with the theoretical relationship.     

The Tiron free radical as a sensitive indicator of chloroplastic photoautoxidation

Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 5–6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and l-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, but added superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.