STEADY-STATE NEAR-INFRARED DETECTION OF SINGLET MOLECULAR OXYGEN: A STERN-VOLMER QUENCHING EXPERIMENT WITH SODIUM AZIDE

Abstract— A sensitive near-infrared detection system incorporating improvements to existing methodologies has been used to characterize the sodium azide quenching of the steady-state luminescence of singlet molecular oxygen at 1270 nm. Stern-Volmer plots which were linear up to 80% quenching of the ¹O₂ generated by rose bengal and eosin Y yielded a rate constant of 5.8 ± 0.1 times 10⁸ M ⁻¹ s⁻¹ for the quenching of ¹O₂ in water, while the rate constants obtained in deuterium oxide with the same sensitizers were 6.28 times 10⁸ M ⁻¹ s⁻¹ and 6.91 times 10⁸ M ⁻¹ s⁻¹ respectively. A flow system minimized the effects of photobleaching of the rose bengal. With a mercury arc light source, the instrument can be used in photosensitization experiments to detect low levels of ¹O₂ production in aqueous media.     

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.     

KINETIC PROPERTIES OF THE TRIPLET STATES OF METHYLENE BLUE AND OTHER PHOTOSENSITIZING DYES

Abstract— The long-lived (> 1 μsec) transients formed in the flash excitation of the representative photosensitizers methylene blue, eosin Y and pyrene have been investigated and various criteria have been used to distinguish between triplet state intermediates and chemical intermediates. Previous assignments of the triplet transients of methylene blue appeared less secure in view of the photochemical reactivity of this dye and its lack of phosphorescence. Earlier assignments of monomeric and dimeric triplet transients of methylene blue are substantiated, however, by the observations that the rate constant for quenching by oxygen is approximately 1/9^th diffusion controlled and the formation rates are commensurate with singlet decay rates and by the observation of triplet-triplet annihilation. Additional evidence in support of monomer triplet assignments for methylene blue and eosin Y is provided by the effect of heavy atom quenchers Cs⁺, Hg²⁺ and T1⁺ on decay rates. Due to chemical reactivity, quenching by I⁻appears less suitable as a diagnostic test for triplet state intermediates. The effect of N₃⁻, which is known to quench singlet oxygen molecules and to alter the course of photosensitized oxidations, on the triplet decay of methylene blue, eosin Y and pyrene is also investigated.     

MIGRATION OF EXCITATION ENERGY FROM THIONINE TO METHYLENE BLUE IN MICELLES

Abstract— The main absorption bands of thionine (Th⁺) and methylene blue (MB⁺) in aqueous solution lie at 598 nm and 664 nm, respectively. This position permits excitation energy transfer from Th⁺ to MB⁺, but not vice versa. We describe here studies of such transfer between these molecules adsorbed on micelles of sodium lauryl sulfate (SLS), imitating, at least to some extent, the state of pigments in chloroplasts.
The SLS concentration was varied from 3.0 to 11 × 10^-3 M. In the presence of dye, aggregation to micelles, each containing 70–100 detergent molecules, begins at about 3.0 × 10^-3 M SLS. Practically all dye ions are adsorbed on these micelles as soon as their formation begins.
Energy transfer from adsorbed Th⁺ ions to adsorbed MB⁺ ions can be demonstrated by observing the quenching of the fluorescence of thionine and the sensitization of that of methylene blue.
At [Th⁺] = [MB⁺] = 1 × 10^-5 M , the most efficient energy transfer (82 per cent efficiency, as derived from measurements of the quenching of Th⁺ fluorescence, or 90 per cent, as derived from sensitization of MB⁺ fluorescence) is observed at the lowest SLS-concentration (3.0 × 10^-3 M ), when the only micelles present are those formed by aggregation of dye-carrying low molecular complexes of SLS with dye cations. Each micelle carries, under these conditions, 10–14 molecules of the two dyes, and the distance between two closest dye ions is about 16 A. Transfer becomes less efficient as the SLS-concentration increases, causing pigment molecules to distribute themselves among a greater number of micelles.     

INCREASED QUANTUM YIELD OF PHOTOREDUCTION OF DYES (A CATALYTIC EFFECT)

Abstract— While studying the photoreduction of some dyes (D) by reducing agents (R), it was observed that the quantum yield of the photoreduction increases considerably upon addition of a third substance (C), whereas it is very small when the dye is photoreduced by C alone (catalytic effect), (see Table 1).
The system thionine (D), allylthiourea (R), and azulene (C) was investigated in detail using both flash photolysis and continuous illumination. On photolysis, thionine is converted into its photo-reduced form, leucothionine. Azulene reacts with the basic form of the thionine triplet ³ TH ⁺ to produce the semithionine radical. In the system thionine and azulene, most of these radicals revert back to thionine. When ATU (˜ 10^2- M ) is added to thionine and azulene (3 × 10^-4 M ), the semithionine radicals are reduced to leucothionine; the quantum yield of this reduction is considerably higher than in the system thionine and allylthiourea. Flash experiments demonstrate that allylthiourea does not react with the semithionine radicals.
At very high ATU concentrations (≥ 10^-1 M ), however, the primary reaction is between thionine triplet and allylthiourea; under these conditions the quantum yield is not influenced by azulene (3 × 10^-4 M ).     

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.     

TRIPLET LIFETIMES OF SOME FLAVINS

Abstract— Using flash photolysis the rate constants of the triplet decay at pH 7.0 (26°C) of 10-hydroxyethyl-isoalloxazine (2700 s⁻¹), 2'-deoxyRF (2300 s⁻¹), 5'-deoxyRF (3200 s⁻¹), 8-hydroxyRF (18 000 s⁻¹) and 8-aminoRF (28 000 s⁻¹) have been measured. The results agree with other evidence that photochemical properties of flavins are influenced by the interaction of the sidechain with the isoalloxazine nucleus. In addition, our data on RF and FMN indicate that the triplet decay rates given in the literature for these compounds have to be corrected to 3200 s⁻¹ (RF) and 4900 s⁻¹ (FMN), respectively. The rate constants for the quenching of the triplet by ground state molecules for all above compounds are given.     

A THEORETICAL TREATMENT OF THE ELECTRONIC STATES OF THIONINE AND RELATED MOLECULES

Abstract— –Problems associated with the protolytic equilibria of thionine and related molecules in their lowest excited electronic states were investigated. The theoretical arguments are based on semi-empirical SCF MO (CI) calculations for the π-electronic system of these molecules; all singly excited configurations were included in the CI. The results indicate that the basic form of thionine in its ground, first excited singlet and lowest triplet state is protonated at the heterocyclic N atom. The difference of the p K values of these three states can be explained in terms of the calculated charge densities. The photochemical reactivity of the lowest triplet of the acidic form of thionine (³TH₂²⁺) differs greatly from that of the lowest triplet of the basic form (³TH⁺). Some arguments for the assignment of nπ* character to ³TH₂²⁺ and ππ* character to ³TH⁺ are advanced.     

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.     

THE SINGLET OXYGEN REACTIVITY OF BILIVERDIN

Abstract— In 1, 1, 2-trichlorotrifluoroethane solution biliverdin physically quenches singlet oxygen at a rate of 8 × l0^sM-¹s^-1 and reacts chemically at 6 × 10 ⁵M-¹s^-1 to give a red product. In D, O solution the rate constants are PD dependent and range from 1.5–6 times 10¹⁰M-¹s^-1 for quenching and the chemical rate varies from 3–5 × 10⁸ M^-1 s^-1 to give colorless products.     

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.     

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.     

The electronic quenching rates of NO(AΣ, v=0–2)

The electronic quenching rates of NO(A²Σ⁺, v^′=0–2) are measured for the gases He, Ar, Xe, N₂, O₂, CO₂, N₂O, and SF₆. The variations of the fluorescence intensity were measured for the (0,0), (1,0), and (2,0) bands of the γ band system when the quencher gases were added. The quenching rates were determined by using the Stern–Volmer plots with the known radiative lifetimes of the excited states. The electronic quenching rate constants are fast for the group of gases of O₂, CO₂, N₂O, and SF₆, whose quenching rate constants are in the order of 10⁻¹⁰ cm³/s. The quenching rate constants are slow for the group of gases including He, Ar, Xe, and N₂ whose rate constants are in the order of 10⁻¹⁴ cm³/s. For the slow group, the quenching rate constants increase rapidly for v^′=2 compared with those of v^′=0 and 1.

The charge transfer model and collision complex model are used to understand the quenching mechanism. For the fast group which mainly consists of gases with positive electron affinities, the charge transfer model adequately describes the mechanism. For the slow quenching group, a theoretical background is provided by consider the coupling of initial and final states in the complex potential surfaces.     

QUENCHING OF CHEMIEXCITED TRIPLET ACETONE BY BIOLOGICALLY IMPORTANT COMPOUNDS IN AQUEOUS MEDIUM

Abstract— Thermolysis of tetramethyl-l,2-dioxetane is a convenient source of triplet acetone, which can be monitored in aerated solutions by the sensitized fluorescence of 9,10-dibromoanthracene. We have investigated the quenching of chemiexcited triplet acetone in air-equilibrated aqueous solutions containing the 9,10-dibromoanthracene-2-sulfonate ion by five classes of compounds: indoles, tyrosine derivatives, quinones, riboflavin, and xanthene dyes. Quenching rates for indoles, tyrosine and its 3,5-dihalogenoderivatives, and xanthene dyes (k_q= 10⁸-10⁹ M^-1 s^-1) are considerably smaller than the diffusion controlled rate, whereas those for quenchers with high electroaffinities, such as quinones (IP = 10–11 eV), approach the diffusion controlled rate (k_q= 10¹⁰ M^-1 s^-1). Energy transfer for riboflavin probably occurs by a triplet-singlet Förster type process.
A comparison of the present data with previous studies of quenching of enzymically generated triplet acetone (isobutanal/O₂/horseradish peroxidase) by the same classes of quenchers (except riboflavin) reveals that, independent of the nature of the quencher and the deactivation mechanism, the Stern-Volmer quenching constants ( k_q t⁰) are systematically about one order of magnitude higher in the enzymatic system. The difference is attributed to a longer lifetime of triplet acetone in the latter case, "protected" in an enzyme cavity against collisions with dissolved oxygen.     

ELECTRON TRANSFER FROM CHLOROPHYLL a TO QUINONE IN MONO- AND MULTILAYER ARRAYS

Abstract— Mono- and multilayers of chlorophyll a (Chl a )– lecithin have been prepared on quartz slides, by means of the Blodgett-Langmuir technique, for fluorescence studies. Self-quenching of the Chl a fluorescence has been observed in Chl a -lecithin single layer excited with a laser light at 632.8 nm. The fluorescence yield is reduced by 50% at a concentration of 7 ± 10¹² Chl a molecules cm⁻². Chl a fluorescence quenching, by adding N,N -distearoyl-1,4-diaminoanthraquinone (SAQ), has been studied. in a single layer, in pure Chl a and also at various dilutions of Chl a in lecithin. The results are explained in terms of a dynamic quenching rather than in terms of a permanent complex formation, at the ground state, between Chl a and SAQ. The fluorescence quenching has been interpreted as the result of an electron transfer from excited Chl a to SAQ, and rate constants of 8.3 ± 10⁻⁵ cm² molecule⁻¹ S⁻¹ and 2.4 ± 10⁻⁴ cm² molecule⁻¹ s⁻¹ have been found for pure diluted Chl a , respectively. Ten per cent of the diluted Chl a fluorescence always remains unquenchable and independent of the quinone concentration. In multilayers, where SAQ and Chl a are in different layers, there is no fluorescence quenching for pure or diluted Chl a even when the chromophores are in two adjacent layers. This happens only if SAQ is not able to diffuse from one layer to another. A minimum value of 22.4 nm has been found for the singlet exciton diffusion length in pure Chl a multilayers.     

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.     

Photosensitizing properties of triplet beta-lapachones in acetonitrile solution

The photochemical reactivity of β-lapachone ( 1 ), nor -β-lapachone ( 2 ) and 1,2-naphthoquinone ( 3 ) towards amino acids and nucleobases or nucleosides has been examined employing the nanosecond laser flash photolysis technique. Excitation (λ = 355 nm) of degassed solutions of 1 – 3 , in acetonitrile, resulted in the formation of their corresponding triplet excited states. These transients were efficiently quenched by l -tryptophan, l -tryptophan methyl ester, l -tyrosine, l -tyrosine methyl ester and l -cysteine ( k _q  ≈ 10⁹ L mol⁻¹ s⁻¹). For l -tryptophan, l -tyrosine and their methyl esters new transients were formed in the quenching process, which were assigned to the corresponding radical pair resulting from an initial electron transfer from the amino acids or their esters to the excited quinone, followed by a fast proton transfer. No measurable quenching rate constants could be observed in the presence of thymine and thymidine. On the other hand, efficient rate constants were obtained when 1 – 3 were quenched by 2'-deoxyguanosine ( k _q  ≈ 10⁹ L mol⁻¹ s⁻¹). The quantum efficiency of singlet oxygen (¹O₂) formation from 1 to 3 was determined employing time-resolved near-IR emission studies upon laser excitation and showed considerably high values in all cases (Φ_Δ = 0.6), which are fully in accord with the ππ* character of these triplets in acetonitrile.     

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.     

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).     

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.