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.     

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.     

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.     

ENERGY CONVERSION IN THE DECAY OF TRIPLET LUMIFLAVIN IN THE PRESENCE OF FERRI- AND FERROCYANIDE

Abstract— Flash photolysis at 450 nm has been used to study the quenching of the excited triplet state of lumiflavin and the transient species formed in subsequent reactions in deaerated phosphate buffer (pH 6.9).
The effect of the presence of ferricyanide on the life time of triplet lumiflavin has been studied. The results suggest an energy transfer reaction without concurrent electron transfer reactions. The rate constant for the process was 2.8 times 10⁹ M ^-1 s^-1. The analogous reaction with ferrocyanide could not be observed because of the efficient electron transfer reaction (δG = -20.6 kcal mol^-1) leading to the formation of the semireduced lumiflavin and ferricyanide. The rate constant for this reaction was 3.3 times 10⁹ M ^-1 s^-1. The semireduced lumiflavin radical was found to disappear in a second order reaction with a rate constant of 1.7 times 10⁹ M ^-1 s^-1. It was found to react with ferricyanide with a rate constant of 0.7 times 10⁹ M ^-1 s^-1.
A model for the various photochemical and photophysical processes involved in the decay and quenching of the lumiflavin triplet state is suggested and discussed.     

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).     

QUENCHING OF TRYPTOPHAN PHOSPHORESCENCE IN ALCOHOL DEHYDROGENASE FROM HORSE LIVER and ITS TEMPERATURE DEPENDENCE

Abstract— The phosphorescence of alcohol dehydrogenase from horse liver (LADH) can be observed at room temperature. The quenching of this long-lived light emission, which comes from a tryptophan residue well buried within the interior of the enzyme structure, was measured. The rate constants for the quenching by the small oxygen molecule and by the I ^-1ion were found to be 1.4 → 10⁸ M ^-1 s^-1 and 10⁸ M ^-1 s^-1, respectively, at room temperature. The temperature dependence of the quenching yields an activation energy of about 14 kcal/mol. This activation energy and the meaning of the accompanying large pre-exponential factor in the Arrhenius equation, A = 10¹⁸ M ^-l s^-1, are discussed in terms of a model in which the quencher threads its way through the protein network.     

MECHANISM OF PHYSICAL QUENCHING OF SINGLET MOLECULAR OXYGEN BY CHLOROPHYLLS and RELATED COMPOUNDS OF BIOLOGICAL INTEREST

Abstract— The rate constant k₅/ > for physical quenching of singlet oxygen O₂(δ₁;) by the sensitizer in dye-sensitized photooxygenations is determined in the case of chlorophylls a and b (7.3 times 10⁸, 4.2 times 10⁸ M^-1 s^-1 respectively), pheophytins a and b (7.4 times 10⁷, 3.0 times 10⁷ M^-1 s^_1 respectively), tetraphenylporphyrin (4.4 times 10⁷ M^-1 s^_1), magnesium tetraphenylporphyrin (5.0 times 10⁸ M^-1 s^_1), zinc tetraphenylporphyrin (1.5 times 10⁸ M^-1 s^_l) and protoporphyrin IX-dimethylester (9.1 times 10⁷ M ^-1 s^_1) in benzene. These sensitizers show a linear correlation between log k_s^O , and their half-wave oxidation potentials and the value of the slope is similar to that observed for various compounds such as phenols. It is concluded that (i) the interaction between chlorophylls and related compounds with singlet oxygen may involve an exciplex as for phenols, and (ii) physical quenching may be envisaged as a spin-orbit-induced intersystem crossing within the exciplex.     

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.     

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.     

USE OF TRIS (2,2'-BIPYRIDINE) RUTHENIUM(II) DICATION IN A NOVEL METHOD FOR THE DETERMINATION OF SINGLET OXYGEN REACTION RATES: APPLICATION TO STUDIES OF GELATIN

Abstract— A novel method for the determination of singlet oxygen reaction rate constants is described and applied to studies of cyclohexadiene in methanol and gelatins in H₂O and D₂O. The technique uses tris (2,2'-bipyridine) ruthenium(II) dication (Ru(bipy)₃₂₊) as both singlet oxygen sensitizer and in situ oxygen concentration monitor during irradiation of sealed samples. Because of the high efficiency with which the luminescence of Ru(bipy)₃2+* can be detected, and the fact that emission lifetimes are used, the method offers some advantages over those previously described. The advantages and disadvantages of the method are discussed. A rate constant of 2.1 (±0.3) x 10⁶ mol^-1 dm³ s^-1 has been determined for the reaction of ¹O₂ with cyclohexadiene in methanol. For two different photographic gelatins the sums of reaction and quenching rate constants are 2.0 (±0.4) x 10⁶ and 3.1 (±2.0) x 10⁵ mol^-1 dm³ s^-1; for swine skin gelatin this value is 3.9 (±2.4) × 10⁵ mol^-1 dm³ s^-1. Chemical reaction, rather than physical quenching, is the dominant reaction route for gelatins and ¹O₂.     

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 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.     

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).     

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.     

DEACTIVATION OF SINGLET MOLECULAR OXYGEN BY THIOLS AND RELATED COMPOUNDS, POSSIBLE PROTECTORS AGAINST SKIN PHOTOSENSITIVITY

Abstract— From time-resolved measurements of the decay of singlet molecular oxygen phosphorescence at 1270 nm in D₂O, direct estimates have been gained for the rate constants of the singlet oxygen reactions with a group of sulphur compounds in the pD range 5 to 13. In the case of most of the thiols, the results are consistent with singlet oxygen reacting exclusively with the thiolate anions. At the normal physiological pH 7, the apparent rate constants (in units of M^-1 s^-1) were 8.9 times 10⁶ (cysteine), 2.5 times 10⁶ (N-acetyl cysteine), 2.9 times 10⁶ (glutathione), 3.0 times 10⁵ (2-mercaptoethanol), 2.3 times 10⁷ (ergothioneine) and 2.7 times 10⁶ (2-mercaptopropionyl glycine). For methionine the rate constant, 1.4 times 10⁷, was independent of pD in the range studied. These sulphur compounds, in particular N-acetyl cysteine and ergothioneine, or related compounds, might be considered as possible candidates for protection against skin photosensitivity side effects associated with the photodynamic therapy of solid tumours and as observed in the disease erythropoietic protoporphyria.     

LUMIFLAVIN-SENSITIZED PHOTOOXYGENATION OF INDOLE

Abstract— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 10⁹ M ^-1 s^-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, ¹O₂. But the reaction between ¹O₂ and indole (7 times 10⁷ M^_l s^_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via ¹O₂ processes, though appreciable ¹O₂ was formed.     

MECHANISM OF CONCENTRATION QUENCHING OF A XANTHENE DYE ENCAPSULATED IN PHOSPHOLIPID VESICLES

Encapsulating a xanthene dye in phospholipid vesicles produces vesicle solutions that contain dye at very high microscopic concentrations, but have a low overall optical density, thereby eliminating reabsorption. Using this system, we have studied the effects of concentration on the fluorescence lifetime of one such dye, sulforhodamine 101. We have observed that the lifetime decreases as a function of encapsulated dye concentration, which is indicative of collisional quenching. The lifetime decreases from 4.5 nsec for sulforhodamine in dilute aqueous solution to 0.69 ns at an encapsulated concentration of 33 m M . The bimolecular rate constant for this event is 2.6 10¹⁰ M ^-1 s^-1, consistent with a diffusion controlled event. However, the quenching constant calculated from changes in intensity is 2.2 10¹¹ M ^-1 s^-1. Thus, collisional quenching is not the predominant mechanism of quenching. The absorption spectra of dye in vesicles indicate an important contribution from static complex formation. Förster distance calculations indicate that energy transfer can also occur to a significant extent, with a predicted efficiency of transfer of 34% at a dye concentration of only 1 m M     

COMPLEX FORMATION BETWEEN ETHIDIUM BROMIDE AND THE NUCLEOTIDES AMP AND GMP

Abstract— The antimicrobial drug ethidium bromide (EB) has been found to form molecular complexes with nucleotides, the strongest being those with the purines AMP and GMP. For the EB-AMP and EB-GMP complexes (which we characterized) the values of the ground-state association constant have been found to be 100 and 103 M ^-1, respectively. The fluorescence of the drug is enhanced when complexed to AMP, GMP, and TMP by about 60, 30 and 10%, respectively, whereas it is quenched by about 20% when complexed to CMP. Fluorescence enhancement analysis has yielded the values of 1.1 × 10¹⁰ and 0.7 × 10¹⁰ M ^-1 s^-1 for the excited-state rate constant of complex formation with the former two nucleotides, respectively. About one out of two collisions between an excited EB molecule and an AMP or GMP molecule results in complex formation. The potential implications of these findings for the EB-DNA interaction are discussed.     

SINGLET OXYGEN YIELDS AND RADICAL CONTRIBUTIONS IN THE DYE-SENSITISED PHOTO-OXIDATION IN METHANOL OF ESTERS OF POLYUNSATURATED FATTY ACIDS (OLEIC, LINOLEIC, LINOLENIC AND ARACHIDONIC)

Abstract— The triplet state characteristics (spectrum, lifetime and quantum yield) for four dye sensi tisers [methylene blue (MB), erythrosin (ER), haematoporphyrin (HP) and riboflavin (RF)] were determined in methanol by laser flash photolysis and singlet oxygen yields (0.60 to 0.48) from time-resolved measurements of the 1270 nm near infrared emission. The reaction of singlet oxygen with four long chain unsaturated phenyl esters [oleate (18: 1), linoleate (18: 2), linolenate (18: 3) and arachidonate (20: 4)] was followed quantitatively using the singlet oxygen luminescence technique and also, after continuous420–700 nm irradiation, by HPLC and other analysis of the isomeric product monohydroperoxides. The overall quantum yield of photooxidation (∼10^-2) was shown to be consistent with the observed singlet oxygen quenching constants(2–12 times 10⁴ dm³ mol^-1 s^-1) for the four esters studied and the singlet oxygen lifetime in methanol (τ∼ 9 μs). The isomer product distribution was interpreted in terms of a dual singlet oxygen and radical mechanism, the radical contribution increasing with sensitiser in the order ER = MB < HP ≪ RF, but also showing some dependence on substrate unsaturation. Evidence is presented for singlet oxygen quenching by MB and RF ( k_O = 1.6 and 6.0 times 10⁷ dm³ mol^-1 s^-1) and for the accelerated photobleaching of the dye sensitisers in the presence of the unsaturated esters.     

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.