PRODUCTS AND RELATIVE REACTION RATES OF THE OXIDATION OF TOCOPHEROLS WITH SINGLET MOLECULAR OXYGEN

Abstract— The relative reactivity of singlet molecular oxygen, 0₂(¹Δ_g), α-,β-,Γ-and δ with -tocopherol (vitamin E) was investigated using microwave discharge generation as a uniquely clean source of singlet oxygen and using a hydrocarbon solvent to approximate the membrane environment. The relative efficiencies of the tocopherols for O₂(¹Δg) were found to decrease in the order: D-α-tocopherol > D-β-tocopheroI > D–Γ-tocopherol > D-δ-tocopherol. The reaction products in all cases were found to be mixtures of quinone and quinone epoxides apparently resulting from decomposition of the primary product, the hydroperoxydienone.     

ON THE PRODUCTION OF NITROXIDE RADICALS BY SINGLET OXYGEN REACTION: AN EPR STUDY

Abstract— The production of free radicals by reaction of 2,2,6,6-tetramethyl-4-piperidinol with singlet oxygen was studied by EPR spectroscopy. The rate constant of the amine was found to be equal to 8 ×10⁵ M ^-1s^-1 in ethanol and to 4 × 10⁷M^-1s^-1 in phosphate buffer (pH 8). Competition experiments were performed with singlet oxygen quenchers such as NaN₃, DABCO and the quenching rate constants were found to be consistent with the literature values. The EPR method proved to be a valuable technique to study the reaction of singlet O₂ with the sterically hindered amine without any interfering effect.     

SOME PREVALENT BIOMOLECULES AS DEFENSES AGAINST SINGLET OXYGEN DAMAGE

Abstract— We have compared the relative abilities of some putative biological protectors to block oxidation of 2,5-bis(hydroxymethyl)furan (BHMF)† in illuminated solutions containing the photosensitizer rose bengal and in the separated-surface-sensitizer (S-S-S) system involving pure singlet oxygen (¹ΔA_gO₂). While L-histidine is a well-known quencher of singlet oxygen, free L-histidine is not commonly found in high concentrations in nature. L-Carnosine (β-alanyl-L-histidine), however, is present in the striated muscles of many organisms, most notably mammals, in concentrations up to 40 m M . At neutral pH, carnosine quenched singlet oxygen more effectively than did equimolar histidine, both in solubilized sensitizer studies and in the S-S-S system. In the pure singlet oxygen system, 1 m M carnosine reduced the rate of BHMF oxidation as effectively as 3 m M histidine alone, or a mixture of 3 mM histidine and 3 m M β-alanine. The fungal product L-ergothioneine (2-thiol-L.-histidine betaine) and its synthetic analogue, 2-thiolhistidine, at 1 m M blocked photosensitized BHMF oxidation using solubilized rose bengal, as did urate at 0.5 m M . All three compounds failed to reduce the rate of BHMF oxidation by singlet oxygen in the S-S-S system, however. Homocarnosine (-γ-aminobutyryl-L-histidine) gave levels of protection against BHMF oxidation identical to histidine, but is present in the central nervous system only at micromolar concentrations. Neither 1 m M imidazole nor 5 m M urea reduced BHMF oxidation in either system. We conclude that some prevalent biomolecules may afford protection either by preventing singlet oxygen production (urate, L-ergothioneine) or by intercepting singlet oxygen once formed (L-carnosine). Such protective devices may be of importance in natural systems.     

THE QUENCHING OF SINGLET OXYGEN BY AMINO ACIDS AND PROTEINS

Abstract— The physical quenching of singlet molecular oxygen (¹Δ_g) by amino acids and proteins in D₂O solution has been measured by their inhibition of the rate of singlet oxygen oxidation of the bilirubin anion. Steady-state singlet oxygen concentrations are produced by irradiating the oxygenated solution with the 1–06 μm output of a Nd-YAG laser, which absorbs directly in the electronic transition ¹Δ_g+ 1 v →³Σ_g^-. The rate of quenching by most of the proteins studied is approximated by the sum of the quenching rates of their amino acids histidine, tryptophan and methionine, which implies that these amino acids in the protein structure are all about equally accessible to the singlet oxygen. The quenching constants differ from those obtained by the ruby-laser methylene-blue-photosensitized method of generating singlet oxygen, or from the results of steady-state methylene-blue-photosensitized oxidation, where singlet oxygen is assumed to be the main reactive species. The singlet oxygen quenching rates in D₂O, pD 8, are (10⁷ℒ mol^-1 s^-1): alanine 0–2, methionine 3, tryptophan 9, histidine 17, carbonic anhydrase 85, lysozyme 150, superoxide dismutase 260, aposuperoxide dismutase 250.     

MECHANISMS OF THE PHOTOSENSITIZED OXIDATION OF TYRAMINE

Abstract— …According to the criteria of enhancement in D₂O and inhibition by sodium azide, the oxidation of tyramine photosensitized by methylene blue is largely a singlet oxygen or Type II process. Its quantum yield approximates 0.3 in D₂O at pH 10. There is a less efficient reaction not quenched by azide, which is assigned to a dye-substrate or Type I process. It gives rise to products with distinct bands at 320 and 285nm. Products of the Type I reaction are further oxidized by singlet oxygen and thereby compete with tyramine for this reagent. Kinetic parameters were estimated by computer simulation of the dependence of quantum yield on extent of reaction. The rate constant for reaction of O₂ (¹Δ_g) with tyramine was estimated to be 2.8 × 10⁸ M ^-1 s ^-1± 20% at pH 10. The reaction was also sensitized by hypericin in what appears to be a Type II process.     

The effect of solvent on tetracene oxidation by singlet molecular oxygen (Δg): aspects of specific solvation

It is evident that the solvent effect on tetracene oxidation by singlet molecular oxygen is predominantly conditioned by a specific solvation of the reaction intermediate in aprotic media that is assumed to be a state with a stressed anionic center.     

ON THE ACRIDINE AND THIAZINE DYE SENSITIZED PHOTODYNAMIC INACTIVATION OF LYSOZYME—SINGLET OXYGEN SELF-QUENCHING BY THE SENSITIZERS

Abstract— The quantum yield of the photodynamic inactivation of lysozyme increases in the sequence acridine orange, methylene blue, proflavine and acriflavine (1:5:6:12). At least up to protein concentrations of 0.1 m M , singlet oxygen is exclusively responsible for the inactivation of the enzyme. For methylene blue, acriflavine and proflavine the quantum yields decrease considerably with increasing dye concentrations. From measurements in H₂O and D₂O buffer solutions it was concluded that in the case of methylene blue the effect is mainly caused by the quenching of singlet oxygen [rate constant (3–4) × 10⁸ M ⁻¹ s⁻¹]. For the acridine sensitizers both singlet oxygen and dye triplet quenching processes have to be taken into consideration. It has been found that all sensitizers act as competitive inhibitors of the enzymatic reaction of lysozyme. However, the dye-protein interaction near the active center cannot be responsible for the observed dye self-quenching effect.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES—VI. PHOTOOXIDATION OF L-TRYPTOPHAN BY SELECTIVELY SULFONATED GALLIUM PHTHALOCYANINES: SINGLET OXYGEN YIELDS AND EFFECT OF AGGREGATION

Abstract— The photosensitized oxidation of L-tryptophan by gallium phthalocyanines sulfonated to different degrees is studied as a function of both substrate and sensitizer concentrations in water and 50% MeOH-H₂O solutions. The maximum quantum yield of singlet oxygen was found to be nearly 0.5 for all sulfonated gallium complexes. The effect of adding sulfonate groups in the phthalocyanine backbone is to change the tendency of dye molecules to dimerize or aggregate in a particular solvent. A shift in the chemical equilibrium away from the monomeric state, which occurs at high dye concentrations and at lower degrees of dye sulfonation, results in a reduced photochemical yield. The variation of quantum yields in different solvent systems and at several wavelengths is similarly accounted for by the fraction of light absorbed by the productive monomer state.     

FLUORESCENCE AND PHOTOOXIDATION OF 1—ANILINONAPHTHALENE—8—SULFONIC ACID IN REVERSED MICELLAR SOLUTIONS AND THE EFFECT OF CCl4

Abstract— The 1-anilinonaphthalene-8-sulfonic acid solubilized in dodecylammonium propionate reversed micellar cyclohexanic solutions, emitted a strong fluorescence, and was photooxidized under aerobic conditions. Carbon tetrachloride (CCl₄) highly quenched the fluorescence and remarkably enhanced the oxidation reaction. The fluorescence quenching obeyed the Stern-Volmer relation, and the photooxidation was caused by the singlet oxygen generated by the photosensitization of the dye. From the kinetic analysis, it was known that the intersystem crossing rate from the dye excited singlet to triplet was enhanced by CCl₄. Carbon tetrachloride did not quench the triplet state. The ratio of quantum yields for the oxidation in the presence and absence of CCl₄ was independent of the oxygen concentration in the reaction mixture. The fluorescence quenching constant and the intersystem crossing rate were obtained at various solubilized water contents.     

Light Emission Resulting from Hydroxylamine-Induced Singlet Oxygen Formation of Oxidizing LDL Particles

Abstract— Oxidation of low-density lipoprotein (LDL) by low amounts of cupric ions resulted in the formation of singlet oxygen (₁O₂, ₁DL_g) when hydroxylamine (NH2OH) was added. Direct evidence on this excited species came from partial spectral resolution of the emitted light in the red spectral region (634 nm and 703 nm), which can be attributed to the dimol decay of singlet oxygen. Additional evidence for the existence of singlet oxygen came from the enhancing effect of deuterium oxide buffer (D20) on chemiluminescence intensity and the quenching effect of sodium azide. A linear correlation between NH2OH-de-pendent chemiluminescence intensity and the amount of diene conjugates (DC) formed in this reaction was observed. Removal of adventitious transition metals by adequate chelators prevented chemiluminescence in this system; NH2OH was also found to efficiently decrease metabolites of lipid peroxidation (LPO). Our findings are consistent with a sequence of reactions in which NH2OH first converts transition metals to their reduced state, thereby stimulating the formation of alkoxy- and peroxy-radicals. Peroxyradicals decompose in a bimolecular Russel reaction to hydroxyl compounds and singlet oxygen while the majority of alkoxy radicals are eliminated by a secondary reaction with NH2OH. Identical effects were observed when reducing antioxidants such as ascorbic acid or trolox C were used instead of hydroxylamine.     

A COMPARATIVE STUDY OF PHOTODYNAMIC OXIDATION AND RADIOFREQUENCY-DISCHARGE-GENERATED 1O2 OXIDATION OF GUANOSINE

Abstract— Guanosine-2-¹⁴C and guanosine-8-¹⁴C were subjected, under similar environmental conditions, to oxidation either by radiofrequency-discharge-generated singlet oxygen or by a methylene-blue-sensitized photodynamic reaction. The reaction products in both cases were compared chromatographically. The product distributions were not identical, indicating differing modes of oxidation in these two systems. It is suggested that both Type I and Type II oxidation mechanisms are operative in the photodynamic system, whereas only Type II-like reactions occur in the radio-frequency-discharge system.     

PHOTOOXIDATION OF 3, 4-DIHYDROXYPHENYLALANINE BY HEMATOPORPHYRIN IN AQUEOUS SOLUTIONS: AN ELECTRON SPIN RESONANCE STUDY USING 2, 2, 6, 6-TETRAMETHYL-4-PIPERIDONE-1-OXYL (TEMPONE)

Abstract— The oxidation of 3, 4-dihydroxyphenylalanine (dopa) photosensitized by hematoporphyrin has been studied by ESR spectroscopy. The nitroxide free radical 2, 2, 6, 6-tetramethyl-4-piperidone-I-oxyl (TEMPONE) is shown to be a convenient probe for monitoring oxygen consumption during the reaction. Isotope and azide effects on oxygen consumption suggest that photooxidation in D₂O proceeds mainly by a singler oxygen mechanism, whereas in H₂O both singlet oxygen and free radical mechanisms are important. The relative importance of these mechanisms can change as oxygen is consumed during the photoreaction. TEMPONE also can be used as an indicator of the presence of reducing radicals (in this case hematoporphyrin radical anions) in the system, but only at low oxygen levels: in aerated solutions efficient reaction of these radical ions with oxygen is proposed.     

PHOTOOXIDATION OF CHLOROPHYLL AND PHEOPHYTIN. QUENCHING OF SINGLET OXYGEN AND INFLUENCE OF THE MICELLAR STRUCTURE

Abstract— When chlorophyll(Chl) and pheophytin(Phn) are irradiated in Triton X-100 water binary solvents, singlet oxygen is formed in the medium in a higher yield for Phn than for Chi. Chlorophyll shows an irreversible photooxidation reaction and a chemical oxidation reaction when 1,3-diphenyliso-benzofuran (DPBF) is added to the solution. During the chemical oxidation, Chi is destroyed by an oxidizing agent that is a reaction product of the endoperoxide formed in the medium by the addition of singlet oxygen to DPBF. This reaction depends on the structure of the medium and has some characteristics of an oxidation by hydroxyl radicals. The highest yield is obtained with the micellar structure. Chlorophyll and Phn are readily oxidized by hydroxyl radicals generated using the Fenton reagent. This suggests that in the presence of Triton X-100, the Mg²⁺ ion of a Chi molecule plays a key role in the irreversible oxidation of the pigment.     

PHOTOOXIDATION OF EPINEPHRINE SENSITIZED BY METHYLENE BLUE—EVIDENCE FOR THE INVOLVEMENT OF SINGLET OXYGEN AND OF SUPEROXIDE

Abstract— The photooxidation of epinephrine, sensitized by methylene blue or by chlorophylls, excited with red light, involves the reduction of two molecules of oxygen to hydrogen peroxide per molecule of epinephrine oxidized to adrenochrome. The initial rates of these reactions are not affected by low concentrations of catalase. In 99 mol % D₂O, rates of methylene blue sensitized photooxidations are accelerated as much as 5.2 times over rates in ordinary water. Azide anion is a more effective inhibitor of this reaction in D₂O than in H₂O. Half maximal inhibitions are obtained by 1.3 mM azide in H₂O and by 0.1 mAf azide in D₂O. Isotope effects and azide sensitivities point to photooxidation of epinephrine in D₂O primarily by a singlet oxygen pathway; in H₂O, non-singlet oxygen pathways become more predominant. Superoxide dismutase (SOD) markedly inhibits rates of the photooxidations in H₂O and in D₂O; about 25% at 10^-9 M SOD, and 50% at 10^-6 M SOD in H₂O. In the photooxidation in H₂O, both by non-singlet and singlet oxygen mechanisms, the amount of superoxide produced is equivalent to the amount of O₂ consumed in the photooxidation of epinephrine; the superoxide thus formed participates in the oxidation of epinephrine.     

Does the DNA Binding Mode of a Molecule Affect its Ability to Interact With Singlet Oxygen?

Singlet oxygen is known to be a potent mutagenic agent and several biologically relevant molecules have been proposed to act as scavengers for this noxious species. However, numerous studies have been conducted in homogenous solution and the reactivity of singlet oxygen scavengers known to bind DNA has never been investigated in double-stranded DNA. In the following paper, we present the results obtained regarding the interaction between 4',6-diamidino-2-phenylindole (DAPI) and singlet oxygen. We show the molecule to be a potent scavenger of singlet oxygen in aqueous solution with an absolute rate constant (chemical and physical quenching of singlet oxygen) of (1.7 ± 0.3) × 10⁷  m ⁻¹ s⁻¹. In addition, we demonstrate that the binding mode of a singlet oxygen scavenger to DNA can strongly influence its reactivity toward singlet oxygen. In the case of DAPI, while the molecule exhibits a chemical reaction with singlet oxygen when the molecule is free in aqueous solution or intercalated in GC sequences of DNA, DAPI becomes chemically unreactive toward singlet oxygen when bound in the minor groove of DNA AT sequences.     

RATE PARAMETERS FOR THE QUENCHING OF SINGLET OXYGEN BY WATER-SOLUBLE AND LIPID-SOLUBLE SUBSTRATES IN AQUEOUS AND MICELLAR SYSTEMS

Abstract— The laser flash photolysis method has been used to determine the bimolecular rate constants for the reaction between O₂(¹Δ₉) and several lipid-soluble and water-soluble substrates. Values for lipid-soluble substrates have been obtained using aqueous dispersions of surfactants above the critical micelle concentration with 1,3 diphenylisobenzofuran as monitor of singlet oxygen. Under these conditions the hydrophobic substances are solubilized by the micellar phase. For substrates which are water-soluble, 9,10-anthracene dipropionic acid disodium salt was used as singlet oxygen monitor. For several substances, the values obtained are comparable to the values found in homogeneous nonaqueous solutions. In cases where significant differences have been found these have been rationalized according to the individual case. The only major unexpected result concerned β-carotene which, in micellar dispersion, failed to react at all with O₂(¹Δ₉) This may be due to multi-molecular aggregations occurring in the polar medium. The work described herein shows clearly that, under appropriate conditions, singlet oxygen kinetics can be effectively followed in aqueous solutions by time resolved methods. The indiscriminate use of β-carotene as a quencher of O₂(¹Δ₉)in mainly aqueous media is questioned.     

AN EXAMPLE OF HOW PHOTOPHYSICAL PROPERTIES CAN INFLUENCE THE MAGNITUDE OF SOLVENT ISOTOPE EFFECTS UPON PHOTO-OXIDATION REACTIONS

Abstract Substantial isotope effects have been observed for the dye sensitised photo-oxidation of 1,3-diphenyl-2-pyrazoline in both polar and non polar solvents, implicating singlet oxygen as a reactive intermediate. By way of contrast, a solvent isotope effect upon the direct photo-oxidation of the pyrazoline was only observed when a protic solvent (methanol) was used. It was found that the photophysical properties (e.g. quantum yields and fluorescence lifetimes) of pyrazolines are sensitive to the isotopic composition of protic solvents but not aprotic solvents. The solvent isotope effect observed for the direct photo-oxidation reaction in methanol may not therefore be a true indication of the participation of singlet oxygen. Since this reaction may not be singlet oxygen mediated, an alternative mechanism is proposed.     

THE ABSOLUTE VALUE OF THE REACTION RATE CONSTANT OF BILIRUBIN WITH SINGLET OXYGEN IN D2O

Abstract— The chemical reaction rate constant of bilirubin with singlet oxygen in basic aqueous solution has been redetermined to be 3.5 × 10⁸ M^-1 s^-1 by a competitive technique using a 1,3-diphenylisobenzofuran in sodium dodecyl sulfate micelles. Bilirubin also physically quenches a singlet oxygen with a rate constant of 9 × 10⁸ M ^-1 s^-1. The lifetime of singlet oxygen in D₂O solution has been determined to be 35 μ s . The absorption cross-section for the molecular oxygen ³∑_g-→¹δ _g + 1 v electronic transition at 1.06μn in aqueous solution is unexpectedly larger than the gas paase cross-section.     

PHOTOSENSITIZED FORMATION OF ASCORBATE RADICALS BY RIBOFLAVIN: AN ESR STUDY

Abstract— The riboflavin-sensitized photooxidation of ascorbate ion (HA^-) to ascorbate radical (A^-) was followed by electron spin resonance (ESR) spectroscopy in conjunction with oxygen depletion measurements. In air-saturated aqueous media, steady-state amounts of A^- are rapidly established upon irradiation. The ESR signal disappears within a few seconds after the light is extinguished–more slowly under constant irradiation as oxygen is depleted. No photooxidation was observed in deaerated media. Similar results were obtained with other flavins and when ascorbyl palmitate was substituted for HA^-. The effect of added superoxide dismutase, catalase, desferrioxamine, and singlet oxygen scavengers (NaN₃ and tryptophan) was studied, as was replacement of water by D₂O and saturation with O₂. The results are indicative of ascorbate free radical production via direct reaction between ascorbate ion and triplet riboflavin in the presence of O₂. While the presence of superoxide ion tends to reduce the steady-state concentration of A^-, competition from the reaction of HA^- with singlet oxygen is less apparent in this system (at HA^-≥ 1 m M ) than in the previously studied aluminum phthalocyanine tetrasulfonate-photosensitized reaction.