LASER PHOTOLYSIS STUDIES OF SUPEROXIDE ADDUCTS OF COBALT(II) AND ZINC(II) PORPHYRINS IN DIMETHYLFORMAMIDE

Abstract— Photochemistry of superoxide adducts of cobalt(II) and zinc(II) porphyrins has been studied by laser photolysis. It was found that the former in dimethlformamide photodissociates the superoxide anion radical, O₂^-, with the quantum yield of 0.5 ± 0.05 at the excitation wavelenths 355 and 532 nm, and the latter gives flurescence and the triple state without giving rise to the photodissociation of O₂^-     

THE ROLE OF O2- IN THE CHEMILUMINESCENCE OF LUMINOL*

Abstract— The chemiluminescence of luminol in buffered aqueous solutions is inhibited by superoxide dismutase. This occurs whether the luminescence is induced by ferricyanide, persulfate, hypochlorite, or by the action of xanthine oxidase on xanthine. Since superoxide dismutase inhibits reactions which involve O₂^-, we conclude that this radical is a constant factor in the chemiluminescence of luminol in aqueous solutions. The kinetics of light production are discussed in terms of hypothetical mechanisms that fit the available data. The strong luminescence of luminol in aprotic solvents or in aqueous systems containing relatively high concentrations of H₂O₂ could not be explored in this way, because superoxide dismutase is inactive under such conditions.     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

LASER FLASH PHOTOKINETIC STUDIES OF ROSE BENGAL SENSITIZED PHOTODYNAMIC INTERACTIONS OF NUCLEOTIDES AND DNA

Abstract— Laser flash photolysis studies of the production of the triplet state of the xanthene dye, rose bengal (RB), have been carried out. The reactions of this state with oxygen to form singlet oxygen and the superoxide anion radical have been observed and yields measured. Quenching of RB(T₁) by oxygen leads to approximately 75% singlet oxygen and 20% superoxide. The reactivity of these species-RB(T₁), O₂(¹Δ_g) and O₂^-—with four nucleotides and DNA have been determined. Only guanine residues showed any noticeable reaction at neutral pH. At higher pH guanine rate constants increased. The consequences to biological photodynamic processes are discussed.     

O2>-DEPENDENT and -INDEPENDENT PHOTOOXIDATION OF QUERCETIN IN THE PRESENCE and ABSENCE OF RIBOFLAVIN and EFFECTS OF ASCORBATE ON THE PHOTOOXIDATION

Abstract— The self-sensitized photooxidation of quercetin was not suppressed by superoxide dismutase (SOD), but suppressed by ascorbate. During the suppression of quercetin photooxidation, ascorbate was oxidized. These results suggest the reduction of oxidized quercetin to quercetin by ascorbate. Quercetin photooxidation in the presence of both riboflavin and EDTA was suppressed by SOD by about 90%. The result suggests the participation of O₂^- in the photooxidation of quercetin. The participation of O₂^- in the quercetin oxidation was confirmed by a xanthine-xanthine oxidase system. Based on these results the physiological and pharmacological functions of quercetin are discussed.     

CALCULATED RATES AND EQUILIBRIA OF THERMAL INTERCONVERSION OF TRIPLET (3g-) AND SINGLET (1Δg and g+) MOLECULAR OXYGEN

Abstract— From spectroscopic data and rate constants in the literature, equilibrium constants and rates of thermal formation of singlet oxygen (¹Δ_g and ¹Σ_g⁺) were calculated for a number of conditions. For the gas phase we estimate K _eq(¹Δ_g³Σ_g^-) = 1.67 exp(-94.31 KJ/RT) and K _eq(¹Σ_g⁺/³Σ_g^-) = 0.33 exp(-157.0 KJ/RT). The calculated rate constants for the ³Σ_g⁺→¹Δ_g transition of O₂ at 25°C varied from 2.5 × 10^-11 s^-1 in water to 4.8 × 10^-16 s^-1 in air, assuming equal solvent interactions with the ground and excited states. Physical quenchers for singlet oxygen are expected to be catalysts for its thermal formation. Equations are presented which allow one to estimate whether such catalysis by quenchers will result in a pro-oxidant effect.     

ON THE GENERATION OF THE HYDROXYLATION AGENT FROM SUPEROXIDE RADICAL. CAN THE HABER–WEISS REACTION BE THE SOURCE OF OH RADICALS?

Abstract— In many biological systems, the role of O₂^- in hydroxylation and toxic processes was assumed to be due to the formation of OH radicals. The Haber-Weiss reaction (Haber and Weiss, 1934)—(H₂O₂+ O₂^-→ OH + OH^-+ O₂) was suggested as the origin of this activity.
In this study it is shown that this reaction pathway is too slow, and that OH is probably formed from the reaction of complexed superoxide with H₂O₂ or/and from the reduction of Fe(III), bound to biological compounds, by O₂^-; the reduced Fe(II) can then react with H₂O₂ as a Fenton reagent, to yield OH.
It is also shown that singlet oxygen cannot be formed in these biological systems neither from the dismutation of OJ nor from the reaction of O₂^- with OH. Singlet oxygen may be formed from the reduction of metal complexes by O₂^-.     

HO2 ELIMINATION FROM α-HYDROXYALKYLPEROXYL RADICALS IN AQUEOUS SOLUTION

Abstract— In aqueous solutions α-hydroxyalkylperoxyl radicals undergo a spontaneous and a base catalysed HO₂ elimination. From kinetic deuterium isotope effects, temperature dependence, and the influence of solvent polarity it was concluded that the spontaneous reaction occurs via an HO₂ elimination followed by the dissociation of the latter into H⁺ and O₂^-. The rate constant of the spontaneous HO₂ elimination increases with increasing methyl substitution in α-position ( k (CH₂(OH)O₂) < 10s^-1 k (CH₃CH(OH)O₂) = 52s^-1 k ((CH₃)₂C(OH)O₂) = 665 s^-1). The OH^- catalysed reaction is somewhat below diffusion controlled. The mixture of peroxyl radicals derived from polyhydric alcohols eliminate HO₂ at two different rates. Possible reasons for this behaviour are discussed. The mixture of the six peroxyl radicals derived from d -glucose are observed to eliminate HO₂ with at least three different rates. The fastest rate is attributed to the HO₂ elimination from the peroxyl radical at C-l ( k > 7000s^-1). Because of the HO₂ eliminations the peroxyl radicals derived from d -glucose do not undergo a chain reaction in contrast to peroxyl radicals not containing an α-OH group. In competition with the first order elimination reactions the α-hydroxylalkylperoxyl radicals undergo a bimolecular decay. These reactions are briefly discussed.     

SEARCH FOR SINGLET OXYGEN LUMINESCENCE IN THE DISPROPORTIONATION OF HO2/O2

Abstract— During the reaction HO₂+ HO₂ (or O₂^-) = H₂O₂+ O₂ in aqueous solution, no luminescence in the region 620–720 nm, expected if the product O₂ were formed in a singlet state, could be detected. If any singlet O₂ is formed, its yield must be less than 10%. Faint luminescence, sometimes found at shorter wavelengths, was shown to arise from reaction of HO₂ with impurities in the reagents present.     

INTERACTIONS OF ASCORBATE AND CHELATED IRON IN A METHYLVIOLOGEN-MEDIATED MEHLER REACTION

Abstract— –In the light, isolated spinach thylakoids consumed O₂ in the presence of methylviologen, and ascorbate was found to interact with this reaction in various ways. Chelating-resin was used to remove metal impurities from the assay medium. Ascorbate diminished the H₂0₂ pool in resin-untreated solutions, while in resin-treated solutions ascorbate had no effect on H₂O₂ concentrations. A Fenton catalyst (Fe-EDTA) increased O₂ uptake in the presence of ascorbate and decreased the amount of O₂ recovered by catalase. Ascorbate tripled the rate of the methylviologen-mediated Mehler reaction, and the O₂ consumed was liberated to 50% of its original concentration by catalase. Superoxide dismutase reversed the effects of ascorbate on the Mehler reaction rates. These results indicate that ascorbate can stimulate Mehler reactions indirectly by promoting a Fenton-type reaction as well as stimulating Mehler reactions directly by reducing 2O₂^- to 2H₂O₂. The promotion of a Fenton-type reaction by ascorbate appears to be the cause of H₂O₂ depletion in resin-untreated solutions.     

THE PHOTOADDITION OF NUCLEOPHILES TO URACIL

Abstract— Quantum yields for 254 nm ultraviolet photoaddition of the nucleophiles hydrazine, HCN, HSO₃^-, methyl amine, and BH₄^- to uracil have been measured; the quantum yields for hydrazine, HCN, and HSO₃^- additions are pH-dependent. The nucleophiles sulfide, azide, chloride, bromide, iodide, nitrite and thiocyanate failed to photo–add under similar conditions. These reactions are interpreted as 1,4-additions to the conjugated enone system of the anti-aromatic compound, uracil; as suggested by S. Y. Wang (Wang and Nnadi, 1968). The nuclear magnetic resonance (NMR) spectrum of the photohydrate of uracil-5-d-showed that the proton was added to the 5-position in a stereochemically random manner. The photoaddition of HSO₃^- takes place at much lower concentrations than required for the thermal addition of this anion and is also stereochemically random.     

THE EFFECT OF IRON ON THE DISTRIBUTION OF SUPEROXIDE AND HYDROXYL RADICALS AS SEEN BY SPIN TRAPPING AND ON THE SUPEROXIDE DISMUTASE ASSAY

Abstract— Using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide we have demonstrated the presence of OH in the xanthine-xanthine oxidase system when iron and/or iron-EDTA (ethylenediamine-tetraacetic acid) is present. With increasing iron (or iron-EDTA) concentration the intensity of the O₂^- spin adduct decreased while that of OH increased. However, use of diethylenetriaminepentaacetic acid (DETAPAC) as a metal chelator in the reaction mixture suppressed the OH spin adduct signal while maintaining the intensity of the signal from the O₂^- spin adduct.
Use of EDTA to eliminate the interfering effects of metal ions in the superoxide dismutase assay employing xanthine oxidase and nitroblue tetrazolium introduces an artifact from the iron present. The interference in the assay from metal ions, including iron, can be eliminated with use of DETAPAC as a metal chelator. Thus, it is possible to make comparisons of measured superoxide dismutase activities even when there are variations in the amount of iron present in the samples.     

ONE-ELECTRON OXIDATION AND REDUCTION OF AZAPROPAZONE AND PHENYLBUTAZONE DERIVATIVES IN AQUEOUS SOLUTION: A PULSE RADIOLYSIS STUDY

Abstract— Using pulse radiolysis techniques, 3 azapropazone and 3 phenylbutazone derivatives all structurally related to the potentially photosensitive anti-inflammatory drug, azapropazone, have been reacted with the free radical oxidants N₃, Br₂^- and (SCN)₂^- as well as with e^-_aq a strong reductant. It is demonstrated that for 5 derivatives, azapropazone (Az), 2-[a-Carboxy-valeryll-3-dimethylamino-7-meth1-1,2-dihydro-1,2,4-benzotriazine (Mi307), phenylbutazone (PB), oxyphenylbutazone (OPB), and ketophenylbutazone (KPB), N₃^- and Br₂^- appear to react via a one-electron removal process. For the other derivative, 8-hydroxy azapropazone (8-OH-Az), Nj and (SCN); oxidise via a one-electron process, while Br₂^- probably fqrms a free radical adduct.
The absolute spectra of the one-electron oxidised and reduced transient species for all six derivatives are thus given in this work and are a basis to the understanding of the action of light on these drugs.     

DETECTION OF OXYGEN RADICALS IN BIOLOGICAL REACTIONS

Abstract— Recent data are presented on the mechanisms or selected assay procedures for superoxide anions (O₂^-) and hydroxyl radicals (OH). The systems discussed include the autoxidation of adrenalin to adrenochrome and other indole compounds, the oxidation of hydroxylamine to nitrite, the generation of ethylene from methional, and the scavenging of OH radicals by p -nitroso-dimethylaniline. The results are compared with other assay procedures to aid in the search for absolute and specific tests for these oxygen radicals. Particular emphasis is placed on the interrelation of 0₂^- OH and hydrogen peroxide.     

DETERMINATION OF SINGLET OXYGEN RATE CONSTANTS IN AQUEOUS SOLUTIONS

Abstract— A very efficient quenching of singlet oxygen (¹O₂) by N₃^- ions has been applied to the determination of rate constants of reactions of ¹O₂ with various substrates (A). This determination has been made possible by choosing experimental conditions which give simple competition between N₃^- and A for ¹O₂ formed in the steady state irradiation of convenient sensitizing dye (S). The consumption of oxygen by the substrate, as followed with an oxygen analyzer, decreases in the presence of low concentrations of N₃^-. Using neutral air saturated aqueous solutions containing the dye phenosafranine + A and varying concentrations of N₃^-, the ¹O₂ rate constants for reactions with biological substrates and some radiation protective agents have been determined.     

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, R_conj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of R_conj by O^- has been found to be more efficient than the less selective OH radical. Peroxidation of R_conj is shown to be somewhat slower, ( k _R+ O₂˜ 3 × 10⁸ M ^-1 s^-1), than O₂ reactions with radicals in oleate ( k _R+ O₂= 1 × 10⁹ M ^-1 s^-1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2 k RO₁˜ 10⁷ M ^-1 s^-1). The superoxide radical, O^-₂, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.     

PROOXIDANT and ANTIOXIDANT EFFECTS OF ASCORBATE ON PHOTOSENSITIZED PEROXIDATION OF LIPIDS IN ERYTHROCYTE MEMBRANES

Abstract— Continuous blue light irradiation of resealed erythrocyte ghosts at 37°C in the presence of uroporphyrin or protoporphyrin results in ¹O₂-mediated (azide inhibitable) lipid peroxidation and membrane lysis. Lipid peroxidation was assessed by thiobarbituric acid reactivity and by quantitation of total hydroperoxides, while lysis was measured in terms of trappedglucose–6-P release. Low concentrations of ascorbate, AH^- (e.g. 0.5 m M ). present at the start of irradiation, significantly enhanced the rates of lysis and peroxidation, whereas relatively high concentrations of AH^- (e.g. 15 m M ) inhibited both processes. By way of contrast. AH^- produced only a dose-dependent inhibition of the photoinactivation of lysozyme, added as an extramembranous target. No significant AH^-induced lipid peroxidation was observed in dark or light controls, plus porphyrin or minus porphyrin, respectively. Stimulation of peroxidation and lysis by low levels of AH^- was enhanced by added Fe(III), abolished by EDTA. but unaffected by catalase or superoxide dismutase. A plausible explanation for these results is as follows. At low concentrations of AH^- prooxidant activity is favored. Redox metal-mediated breakdown of photoperoxides occurs, which tends to amplify lipid peroxidation. Neither O₂^- nor H₂O₂ appears to be involved. At significantly high concentrations, AH^- acts predominantly as an antioxidant by intercepting ¹O₂ and/or sensitizer triplet, or by scavenging free radical intermediates of lipid peroxidation.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

PHOTOFRAGMENTATION OF α-OXOCARBOXYLIC ACIDS IN AQUEOUS SOLUTION. AN EPR STUDY. FORMATION OF SEMIDIONE RADICALS BY DECARBOXYLATIVE SUBSTITUTION OF α-OXOCARBOXYLIC ACIDS BY ACYL RADICALS-I: GLYOXYLIC, PYRUVIC, α-OXOBUTYRIC, α-OXOGLUTARIC AND α-OXOISOCAPROlC ACIDS

Abstract— By means of in situ photolysis EPR of aqueous solutions of α-oxocarboxylic acids (RCO-CO₂H) at pH values above 5, semidione radical anions [RC(O^-)=C(O')R] and α-hydroxy-α-carboxy alkyl radicals [RC(OH)CO₂-] were detected. C0₂ was identified as a reaction product. On photolysis of mixtures of α-oxocarboxylic acids (RCOCO₂H and R'COCC₂H), "mixed" semidione radical anions [RC(O^->=C(O)R'] were observed in addition to RC(O^-)=C(O')R, R'C(O^-)=C(O')R', RC(OH)CO₂^- and R'C(OH)CO₂^-. The experimental results are explained in terms of photodecarboxylation (α-clea-vage) of electronically excited RCOCOJ to yield RCO and CO₂. The radicals RC(OH)CO₂^- are formed by reduction of RCOCO₂^- by CO₂^-. The semidione radicals are produced by addition of RCO to RCOCO₂^- followed by decarboxylation of the intermediate adduct. This mechanism was confirmed by generating acyl radicals independently and reacting them with α-oxocarboxylic acids. Selected product studies support the mechanism suggested.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.