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.     

SYNTHESES AND PHOTOREDOX PROPERTIES OF POLYELECTROLYTES FUNCTIONALIZED WITH 5-DEAZAFLAVIN

Abstract— Anionic polyelectrolytes functionalized with the 5-deazaflavin group (dFl) were synthesized. The lifetime of the triplet excited dFl in the polyelectrolytes with a 2-mol% dFl content (AdFl-2) was about 10 times longer than that of a low molecular weight analog (AdFl-M). 2-Mercaptoethanol (RSH) reduced the triplet dFl with the rate constant of k _red= 2.01 × 10⁸ M ⁻¹ s⁻¹ for AdFl-M and k _red= 4.4 × 10⁷ M ⁻¹ s⁻¹ for AdFl-2. A zwitterionic viologen (SPV) oxidized the triplet dFl with the rate constant of k _red= 3.69 × 10⁹ M ⁻¹ s⁻¹ for AdFl-M and k _ox= 7.4 × 10⁸ M⁻¹ s⁻¹ for AdFl-2. The smaller rate constants for the polymer system were discussed in terms of the hindering effect of the macromolecular microenvironment. The back electron transfer was shown to be drastically slowed in the AdFl-2-SPV system as a result of the intensive electrostatic effect of the polyelectrolytes. The buildup of the viologen radicals was studied under the steady-state illumination of the three component systems including viologen and RSH. The dFl group was demonstrated to serve as a very efficient photosensitizer in the oxidative cycle in case back electron transfer was retarded. This is the case of the AdFl-2-SPV system which gave the quantum yield of about 0.4 for the SPV⁻ buildup. By comparison, the AdFl-2-MV²+ system resulted in a much slower buildup of MV +radicals.     

CHLORIDE ENHANCEMENT OF QUENCHING OF TRIPLET METHYLENE BLUE BY Fe(II)

Abstract— The influence of chloride ion on the rate of decay of triplet methylene blue in 0.01 M acid in the absence and presence of ferrous ions was investigated by means of laser flash-photolysis monitored by kinetic spectrophotometry. Chloride weakly accelerates decay of ³MBH^2± in aqueous solution in the absence of Fe(II). Quenching of ³MBH²⁺ by Fe(II) is more strongly catalyzed by Cl^- in both water and 50 v/v% aq. CH₃CN. The uncatalyzed quenching constant, k ₅, is of the order of 1 × 10⁶ M ^-1 s^-1 while in 4.8 M aqueous chloride ( μ – 7.2 M ) k ₅= (37.2 ± 1.8) × 10⁶ M ^-1 s^-1. A possible role of chloride is as a bridging species in quenching via electron transfer between ³MBH²⁺ and Fe(II).     

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.     

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.     

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.     

THE INFLUENCE OF MOLECULAR CONFORMATION ON THE STABILITY OF ULTRAVIOLET STABILIZERS TOWARD DIRECT AND DYE-SENSITIZED PHOTOIRRADIATION: THE CASE OF 2-(2'-HYDROXY-5'-METHYPHENYL)BENZOTRIAZOLE (TIN P)

Abstract— The mechanism for photodegradation of the ultraviolet photostabilizer 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (TIN P) upon direct and dye-sensitized (singlet molecular oxygen [O₂(1Δ_g)]-mediated) irradiation was studied. From the experimental TIN P photodegradation rate data, and low temperature (77 K) fluorescence and phosphorescence quantum yields, one can conclude that the photodegradative process involves phosphorescent states of TIN P. The open conformer of TIN P quenches O₂(¹Δ_g) by physical scavenging with a rate constant (k_q) in dimethylsulfoxide of 2.8 times 10⁶ M ^-1 s^-1. The intramolecular hydrogen-bonded conformer does not appreciably interact with O₂(¹Δ_g). In the presence of a relatively high concentration of OH- (either 5 times 10^-2 M KOH in ethanol or water at pH 13), the ionic form of TIN P (with an ionized phenol group) physically and chemically quenches O₂(¹Δ_g). The reaction rate constant ( k _r) is 1 times 10 ⁸ M ^-1 s^-1, and the ratio k _q/ k _r is approximately three in alkaline aqueous media.     

Photosensitized reduction of methyl viologen using eosin-Y in presence of a sacrificial electron donor in water-alcohol mixture

Photoreduction of methyl viologen (MV²⁺) by eosin-Y (EY²⁻) in the presence of triethanolamine (TEOA) has been investigated in water–methanol mixture by means of steady-state photolysis and laser-flash photolysis in the visible/near-infrared regions. The complete conversion to the persistent methyl viologen radical cation (MV^·+) was observed in the presence of lower concentrations of EY²⁻ and excess TEOA. By laser-flash photolysis measurements, electron transfer was confirmed to occur from the triplet state of EY²⁻ [³(EY²⁻)*] to MV²⁺ in the rate constants of ca 2.0 × 10¹⁰ M ⁻¹ s⁻¹. The rates and efficiencies of production of MV^·+ were found to be dependent on solvent compositions and concentrations of MV²⁺ ionic salt and TEOA. The back electron transfer reaction from MV^·+ to EY^·− was retarded in the presence of TEOA, which supports that EY²⁻ is reproduced by accepting an electron from TEOA. In the presence of excess TEOA, the indirect formation of MV^·+ from EY^·3− which was produced by accepting an electron from TEOA, was confirmed. The contributions of both the oxidative and reductive routes of ³(EY²⁻)* for the MV^·+ formation have been confirmed.     

THE TRIPLET STATE OF 8-METHOXYPSORALEN

Abstract— Transient absorption spectra produced by laser flash photolysis of an aqueous solution of 8-methoxypsoralen (8-MOP) have been studied. The biphotonic production of hydrated electrons and of the radical ions, 8-MOP ⁺ and 8-MOP^- is reported. The hydrated electron was found to react with ground state 8-MOP with k ˜ 3 × 10¹⁰ M ^-1 s^-1. In order to obtain a true triplet-triplet absorption spectrum. contributions from the radical ions were subtracted from the overall transient absorption. In addition, contributions from e^-_aq to the transient spectrum were removed by using N₂O, low laser intensity to minimize photoionization or by measuring the transient O.D. after the electron has decayed. These three methods each produced the same triplet-triplet spectrum which differs in the red region from previously reported spectra.     

GAMMA AND PULSE RADIOLYSIS STUDIES OF THE REACTION BETWEEN SUPEROXIDE IONS AND OXYHEMOGLOBIN-METHEMOGLOBIN SYSTEM

Abstract— The radiolytic studies of oxyhemoglobin or methemoglobin in neutral aerated aqueous solutions with formate ions, lead to three conclusions:
The oxidation of oxyhemoglobin by O^-₂ is not important. The observed low oxidation yield is probably due to the slow reaction with hydrogen peroxide produced by O^-₂ disproportionation.
The reduction of methemoglobin in γ radiolysis reaches a plateau which could be explained by structural considerations.
The reduction of methemoglobin by O^-₂ ions, if it occurs, is relatively slow: k = 1.4 × 10³ M ^-1 s^-1. But a problem remains concerning the spectral characteristics of the product.     

ON THE REACTION OF SINGLET MOLECULAR OXYGEN WITH N-ALLYLTHIOUREA IN AQUEOUS SOLUTION

Abstract— Experiments on the photooxidation of N -allylthiourea, thiourea, and N-allylurea sensitized by the dye phenosafranine show that in N -allylthiourea the thiourea group is the site of singlet oxygen attack, while the allyl moiety neither reacts with nor quenches this metastable form of O₂ (in neutral aqueous solutions). Low concentrations of N^-₃ (a known quencher of singlet oxygen) strongly reduce the photooxidation of allylthiourea by a mechanism which apparently obeys simple competition kinetics. From these results the rate constant of the reaction between allylthiourea and singlet oxygen is obtained ( k = 4 × 10⁶ M ^-1 s^-1; pH = 7.1).     

MESODIPHENYLHELIANTHRENE—THE MOST REACTIVE SINGLET OXYGEN ACCEPTOR

Abstract— The self-sensitized photooxidation of mesodiphenylhelianthrene in various solvents has been investigated. The involvement of ¹O₂ as the reactive intermediate in the formation of the endoperoxide has been demonstrated by the quenching of the photooxidation by the efficient ¹O₂-quencher β-carotene. The rate constant for the addition of ¹O₂ to mesodiphenylhelianthrene has been determined to be k _R≅ 10¹⁰ M ^-1 s^-1, which is the highest value hitherto known in the literature. The probability factor p , which describes the concentration independent part of the overall quantum yield, has been measured to be p =0.17.     

THE PHOTOOXIDATION OF DIETHYLHYDROXYLAMINE BY ROSE BENGAL IN MICELLAR AND NONMICELLAR AQUEOUS SOLUTIONS

The photooxidation of N,N -diethylhydroxylamine (DEHA) by Rose Bengal (RB) has been investigated in micellar and nonmicellar aqueous solutions. We measured the quantum yield of oxygen consumption forming H₂O₂ and monitored two intermediates, the superoxide and diethylnitroxide radicals. When the pH was vaned, the quantum yield of oxidation remained constant for 6 < pH < 10.5, decreased in acidic pH, and increased considerably in NaOH solution; these changes could be attributed to the protonation and dissociation processes of the >N-OH moiety of DEHA. The formation of diethylnitroxide radical was enhanced by superoxide dismutase or strong alkaline solution. Around neutral pH, the oxidation proceeded mainly via electron transfer from DEHA to the RB triplet ( k _q = 10⁷ M ^-1 s^-1) with little ¹O₂ participation ( k_q < 10⁵ M ^-1 s^-1). However, when RB was incorporated into micelles in alkaline solution, the contribution of the singlet oxygen pathway increased at the expense of electron transfer, which was inhibited by the less polar micellar environment. Dark autoxidation of DEHA was accelerated by heavy metal impurities and increased very strongly in NaOH solution.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: SECONDARY OXIDATION OF REDUCED VIOLOGEN ACCEPTORS BY Ru(NH3)63+

Abstract— The kinetics of the oxidation of a homologous series of 4,4'-di(n-alkyl)-bipyridinium (viologen) radicals by Ru(NH₃)₆³⁺ in vesicle suspensions was studied using laser flash photolysis. The viologen radicals were produced photochemically in the bilayer membrane phase of the vesicles by electron transfer from the triplet state of chlorophyll-α. At high concentrations of Ru(NH₃)₆³⁺, the rate of oxidation of the viologen radicals in the aqueous phase was limited by the rate at which the radicals diffused from the membrane to the aqueous phase. The exit rate constant decreased from 2 × 10⁵ s⁻¹ for the methyl viologen radical to 4 × 10³ s⁻¹ for the pentyl viologen radical. Both the exit rate constants and the calculated values for the equilibrium association constants of the viologen radicals were unexpectedly insensitive to the length of their alkyl substituents. This, as well as other data, suggests that the radicals that diffused into the aqueous phase tended to remain associated with the membrane-water interface.     

Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract— The dynamics of the flavin bound to the flavocytochrome b₂ from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13-fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 10⁹ M ^-1 s^-1 when the flavin is bound to the enzyme and 8.3 × 10⁹ Mv s^-1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b₂ is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.     

ELECTRON TRANSFER FROM THE EXCITED STATE OF TYROSINE TO COMPOUNDS CONTAINING DISULFIDE LINKAGES

Abstract— The flash photolysis of aqueous solutions of tyrosine has been studied in the presence of various concentrations of the cyclic disulfide sodium lipoate (thioctic acid, Na⁺ salt). In addition to the formation of phenoxyl radicals and hydrated electrons (and possibly H atoms) from the photoionization of tyrosine, the characteristic spectrum of the radical anion RSSR^- of lipoate was also observed in neutral as well as in alkaline solutions. From the dependence of these yields upon the concentration of lipoate, it was found that a long–lived triplet excited state of tyrosine, rather than the singlet excited state, is involved in these reactions. The negative radical ions RSSR^- are formed by two distinct pathways: (a) Na⁺–lipoate reacts with the solvated electrons which are ejected from the tyrosine triplets ³Tyr → RO.+ e ^-_aq+ H⁺ followed by e ^-_aq+ RSSR → RSSR^-, and (b) by direct interaction of lipoate with triplet excited tyrosine, resulting in the transfer of a negative charge from tyrosine to the disulfide linkage. At high lipoate concentrations, the singlet excited state of lipoate is quenched, k ₄= 1.6 × 10¹⁰ M ^-1 sec^-1, but this reaction does not lead to the formation of RSSR^- radical ions.     

ASCORBIC ACID AS A PHOTOOXIDATION INHIBITOR

Abstract— The rate constant for total quenching of singlet oxygen by ascorbic acid has been determined using the inhibition of the bleaching of 9, 10-dimethylanthracene by AA in pyridine. The rate constant was 8.4 × 10^-6 M ^-1 s^-1, as determined photochemically, and 1.06 10^-7 M ^-1 s^-1 as determined in a dark reaction.     

TRANSIENT PHOTOCHEMISTRY OF NEUTRAL RED

Abstract— Flash photolysis of neutral red between pH 1.3 and pH 11 yields the triplet species ³DH₂⁺²³DH⁺ and ³D. Both ³DH₂⁺² and ³D exhibit first order decay with rate constants of 1.6 ± 0.3 × 10⁴ s^-1 but ³DH⁺ decays within the lifetime of the flash. Over the entire pH range, ascorbic acid quenches the triplet, forming the semireduced radicals DH₃⁺² DH₂⁺ and DH, all of which exhibit second order decay with k = 1.8 ± 0.4 ± 10⁸ M ^-1s^-1 most probably by recombination with semioxidized ascorbic acid. The dependence of the rate of decay of radical neutral red on the identity of reversible reductants supports the back-electron transfer mechanism, as does digital simulation of complex radical disproportionation schemes. In contrast to the efficient reduction of triplet neutral red by ascorbic acid, its reduction by EDTA is quite inefficient.     

RATES OF REACTION OF SINGLET OXYGEN WITH SULFIDES

Abstract—Reaction rate constants for the reaction of singlet oxygen with a series of 24 sulfides in chloroform have been measured by inhibition of the self-sensitized photooxidation of rubrene. The reaction rate constant is sensitive to steric effects, decreasing as the carbons α- to sulfur become more highly substituted. Addition of a methyl group to each of the carbons α- to sulfur decreases the rate constant by about a factor of 10. From a series of p - and m -substituted thioanisoles, a ρ of -1.67 ± 0.09 was found. A much better correlation was found with σ than with σ⁺ indicating there is no resonance interaction with the reaction center. Typical rate constants are: di- n -butyl sulfide, 2.3 × 10⁷ M ^-1 s^-1; CBZ-L-methionine methyl ester, 1.4 × 10⁷; di-s-butyl sulfide, 1.8 × 10⁶; di- t -butyl sulfide, 1.3 × 10⁵; and thioanisole, 2.3 × 10⁶.     

QUENCHING OF EXCITED TRIPLET LUMIFLAVIN BY LUMIFLAVIN RADICALS FORMED IN ITS T-T REACTION

Abstract— Reactions of the triplet state of lumiflavin (³LF) in water adjusted at pH 7.2 were reexamined by means of a Xe-flash photolysis and a laser photolysis. Measurements of the decay of ³LF were made on solutions of LF ranging in the concentration from 4 to 61 times 10^-6 mol/dm³. A one-electron reduced and a one-electron oxidized species of lumiflavin (LF^- and LF⁺) were produced in the first decay stage of ³LF with a high efficiency (0.6 ± 0.1) in a bimolecular triplet-triplet reaction. The product radicals (LFH^- and LF⁺) quench ³LF very efficiently (3 ± 0.8 × 10⁹ mol^-1dm³ s^-1) compared with LF in the ground state (> 2 × 10⁷ dm³ mol^-1).