DECAY KINETICS OF THE TRIPLET EXCITED STATE OF LUMIFLAVIN

Abstract— The rate of decay of the triplet state of lumiflavin in deaerated phosphate buffer at pH 6.9, has been studied using the flash photolysis technique, at various concentrations and flash energies. The decay of the transient appears as a mixture of first and second order processes, the relative importance of which depends on the experimental conditions.
The following competitive reactions have been found to explain the course of the reaction of the triplet excited state of lumiflavin.     

A FLASH PHOTOLYSIS STUDY OF THE TRIPLET STATE OF ACRIDINE ORANGE IN BASIC SOLVENTS

Abstract— Photochemical reactions of acridine orange (AO) in basic aqueous and ethanolic solutions were studied using the flash photolysis technique. The absorption spectrum of AO was determined in detail (230–900 nm) and extinction coefficients were obtained. The decay of the triplet state is the result of a first order process, a triplet-triplet annihilation process and a quenching by the dye in the ground state. The main part of the triplet decays to the ground state; however the observation of semi-reduced AO shows that the decay is partly due to chemical reactions.
An efficient reversible reaction is observed on flashing aqueous solutions of AO containing tetramethyl- p -phenylenediamine: semi-reduced AO is formed in high yield by reaction between the triplet dye and the diamine. In addition, irreversible reactions of AO occur; these are shown to be due to the triplet state by the method of triplet energy transfer.     

THE TRIPLET STATE OFPYRIDOXAL–5'-PHOSPHATE IN LASER FLASH PHOTOLYSIS

Abstract— Sensitization and quenching experiments demonstrate that laser flash photolysis ofpyridoxal–5'-phosphate populates a triplet state about 244 kJ/mol. Excitation by the 337.1-nm photons from a nitrogen laser generated transient absorption in the 400-nm region which decayed with a fast first order component on a much slower one. Xanthone photosensitized the same absorption; oxygen, nitric oxide, biacetyl, and 1,3-cyclo-hexadiene quenched it. Spectra and lifetimes suggest that the triplet state leads to radical formation.     

PHOTO-EXCITED TRIPLET STATE OF DIMERIC CHLOROPHYLL a IN 3-METHYLPENTANE RIGID MATRIX AT 77 K STUDIED BY FLASH PHOTOLYSIS

Abstract— Flash photolysis studies of dimeric and monomeric chlorophyll a were carried out at 77 K. The triplet-triplet absorption spectrum of the dimeric chlorophyll a in 3-methylpentane at 77 K is interpreted as a sum of the spectra of chlorophyll a in the ground and triplet states. The dimeric chlorophyll a in the triplet state is considered to have the half-excited structure at 77 K without photodisaggregation owing to high viscosity of the solvent.     

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).     

THE EFFECT OF 8α-SUBSTITUTION ON FLAVIN TRIPLET STATE AND SEMIQUINONE PROPERTIES AS INVESTIGATED BY FLASH PHOTOLYSIS*

Abstract— Flash photolysis techniques have been used to study the effect of 8α-substitution on flavin triplet state formation and decay and on the properties of neutral and anionic serniquinones. Compared with riboflavin, the N(1) and N(3) isomers of 8α-histidylriboflavin show a lower triplet yield (?10%) and a faster rate of decay (? 4-Cfold). Acetylation of the histidyl a-amino groups and of the flavin ribityl side chain results in a 2-fold increase in triplet yield and a 2-fold slower rate of decay. The yield of neutral 8α-substituted flavin semiquinones upon flash photolysis in the presence of EDTA was approximately 50% that given by riboflavin. These substituted flavin neutral semiquinones dismutated at a rate 2–3 times slower than the corresponding unsubstituted form, although the anionic semiquinones dismutated at approximately the same rate. In the presence of oxygen, the kinetics of semiquinone decay changed from second order to pseudo-first order upon raising the pH, thus showing anionic semiquinone oxidation as seen previously with unsnbstituted flavins. The pK values for the ionization of the neutral 8α-substituted Aavin semiquinones are 1–1.5 units lower than the unsubstituted form. The anionic 8α-substituted flavin semiquinones react with oxygen at a rate 2–10 times more slowly than does the riboflavin form. Such alterations in properties probably reflect the electron-withdrawing effect of the 8α-substituents on the flavin ring system.     

POTENTIOMETRIC and LASER FLASH PHOTOLYSIS STUDIES OF THE pH DEPENDENCE OF HYDROGEN PEROXIDE PRODUCTION BY THE SEMICARBAZIDE/LUMIFLAVIN/OXYGEN PHOTOSYSTEM

The effect of pH on hydrogen peroxide photoproduction by the semicarbazide/lumiflavin/ oxygen system has been investigated by using the potentiometric and laser flash photolysis techniques. Kinetic analysis of (a) primary and secondary reactions involved in the photoreduction of lumiflavin and (b) lumiflavin reoxidation by oxygen have been carried out at varying pH values, drastic differences being observed in the pH dependence of both types of reactions. The reaction mechanism appears to proceed by semicarbazide-quenching (reductive type) of the light-excited lumiflavin in its triplet state followed, depending on pH, either by reoxidation of the so formed flavosemiquinone by molecular oxygen or by flavosemiquinone disproportionation into oxidized and fully reduced lumiflavin.     

A STUDY OF THE PHOTOCHEMICAL PROPERTIES OF SOME CINNAMATE SUNSCREENS BY STEADY STATE AND LASER FLASH PHOTOLYSIS

The E ⇄ Z photoisomerization of 4'-methoxycinnamates, used as sunscreens in cosmetics, has been studied by steady state and laser flash photolysis, in aqueous and organic solutions. Photoisomerization quantum yields are found to be fairly high (˜0.5-1), although no intermediate is detected upon laser flash photolysis. Cinnamates are not photodynamic sensitizers but are able to quench the 8-methoxy-psoralen and 5-methoxypsoralen triplets which produces E → Z isomerization.     

THE CHARACTERISATION OF THE TRIPLET STATE OF CROCETIN, A WATER SOLUBLE CAROTENOID, BY NANOSECOND LASER FLASH PHOTOLYSES

Abstract— The triplet state of crocetin, which is a water soluble carotenoid, has been sensitized by psoralen. The triplet extinction coefficient, ε_T (73000 dm³ mol^-1 cm^-1 at 470 nm), the triplet-triplet spectrum and the quantum yield of triplet formation, φ_T (less than 1%) are reported in aqueous solution.
In order to calculate the extinction coefficient of crocetin it was necessary to obtain ε_T for psoralen in water (10000dm³ mol^-1 cm^-1 at 450 nm). This latter value was obtained using the complete conversion technique and is reported with the triplet-triplet spectrum.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

INDICATION OF FAST TRIPLET FORMATION IN BIMOLECULAR QUENCHING OF EXCITED SINGLET STATES STUDIED BY CONVENTIONAL FLASH PHOTOLYSIS

The bimolecular quenching of the first excited singlet state of oxonine by allylthiourea leads to the formation of the triplet state of the dye. This has been proved by comparison with the triplet-triplet absorption spectrum of oxonine obtained by triplet-triplet energy transfer. The conventional flash experiments suggest that the dye triplet state is produced directly rather than by radical recombination.     

THE PHOTOREDUCTION OF METHYLENE BLUE BY AMINES—I. A FLASH PHOTOLYSIS STUDY OF THE REACTION BETWEEN TRIPLET METHYLENE BLUE AND AMINES

Abstract— Flash photolysis was used to study the reduction of the triplet state of methylene blue by both alkyl- and aryl-amines. The extent of the formation of the semireduced form of the dye yielded rate constants of interaction between the triplet state and the amine ( k ₅). A correlation between log k ₅ and ionization potentials for alkylamines (slope = -1.75 eV^-1) was interpreted as evidence for the formation of a partial charge-transfer intermediate. The rate constants ( k ₅) calculated for aryl-amines approached the rate of diffusion in many cases. A Hammett plot for a series of N, N-dimethyl-anilines yielded a moderately large p value (– 3.28) consistent with the formation of a charge-transfer intermediate. It was concluded that reaction of amines with triplet methylene blue leads to the formation of a partial charge-transfer intermediate which may undergo complete electron transfer to yield radicals, or undergo spin inversion and return to the ground state.     

THE STEPWISE BIPHOTONIC PHOTOIONIZATION OF CHLORPROMAZINE AS SEEN BY LASER FLASH PHOTOLYSIS

It is generally accepted that both promazine (PZ) and chlorpromazine (CPZ) photionize monophotonically to their respective cation radicals and the corresponding hydrated electrons. It is also supposed that this photoinization has a role in the phototoxic effects of these drugs. However, using laser flash photolysis, we have observed that photoionization of CPZ during S1 excitation (lambda greater than 300 nm) is a stepwise biphotonic process. In the case of PZ our flash photolysis results are less clearcut, but are consistent with stepwise biphotonic photoionization for S1 excitation. We demonstrate, using computer simulation of the intramolecular kinetics, that the estimated triplet state lifetime of CPZ is sufficiently long (23 ns at room temperature) to account for the apparent monophotonic photoionization that has been observed by others at high light intensities and short pulse times. Our laser flash photolysis results also suggest that the photo-ionization mechanism of PZ and CPZ is wavelength-dependent. Both drugs exhibit apparent monophotonic photoionization when they are excited at 266 nm under conditions of laser pulse width and intensity similar to those at 355 nm. We suggest that photoionization is not an important mechanism in the observed phototoxic and photoallergic effects of PZ and CPZ in sunlight.     

TRIPLET EXCITATION TRANSFER INVOLVING β-IONONE. A KINETIC STUDY BY LASER FLASH PHOTOLYSIS

Abstract— Employing nanosecond laser flash photolysis, β-ionone (BI) has been examined as an acceptor and as a donor of triplet excitation. In the limit of diffusion-control as well as below it, the rate constants for the quenching of a series of sensitizer triplets by BI are2–3 times smaller than those by 2,4-hexadienal (HD), although the triplet energies (spectroscopic) of the two carbonyl-containing dienes are estimated to be the same (∼55 kcal mol^-1). We attribute the difference to a steric effect arising from ground-state geometric distortion and heavy alkyl-substitution in BI. In spite of possible exothermic energy transfer, BI triplet is nearly nonquenchable by azulene and ferrocene; this is explainable by torsional relaxation to an equilibrium geometry at which the vertical energy gap is smaller than 40 kcal mol^-1. The singlet oxygen yield from the interaction of BI triplet with oxygen in benzene is estimated to be 0.5, suggesting that spin-exchange and energy-transfer may be involved to the same extent in the oxygen quenching process.     

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.     

A LASER FLASH PHOTOLYSIS STUDY OF THE NATURE OF FLAVIN MONONUCLEOTIDE TRIPLET STATES AND THE REACTIONS OF THE NEUTRAL FORM WITH AMINO ACIDS

Abstract— Primary photochemical processes in aqueous solution have been characterised for FMN. The influence of pH on these processes is attributed to protonation of the neutral triplet and not to the presence of a dimeric species as postulated earlier. Second order rate constants for reaction between the neutral triplet and some naturally occurring amino acids are reported.     

THE ANAEROBIC PHOTOLYSIS OF LENS α-CRYSTALLIN: EVIDENCE FOR TRIPLET STATE MEDIATED PHOTODAMAGE

Anaerobic solutions of lens alpha-crystallin were subjected to near-UV (greater than 295 nm) irradiation, and the photoproducts were analyzed by fluorescence and room-temperature phosphorescence spectroscopy. The principal photoproduct was excited maximally at 340 nm, fluoresced maximally at 430 nm, and phosphoresced with an emission maximum at 510 nm. The phosphorescence intensity decay of this species was well fit by a sum of two exponentials with lifetimes of 9.2 ms (78%) and 61 ms (22%); this report is the first demonstration of a long-lived triplet state associated with a protein photolysis product. As reported previously, 3trp* is also long-lived in deoxygenated alpha-crystallin solution at room-temperature (Berger and Vanderkooi, 1989, Biochemistry 28, 5501-5508), hence both tryptophan and photoproduct triplet states are good candidates to mediate photodamage. Photolysis experiments in the presence of agents known to alter the tryptophan triplet yield provide evidence for the importance of triplet-state-mediated photodamage of lens crystallins in anaerobic solution. In 30 mM acrylamide where 3trp*, but not 1trp*, is efficiently quenched, anaerobic solutions exhibited marked resistance to protein photodamage, whereas the photoprotection in aerobic solution was minimal. In D2O, where photoionization is suppressed but triplet states are longer-lived, photodamage was accelerated in anaerobic solution but reduced in aerobic solutions. Finally, the anaerobic photodestruction rate was increased in 500 mM Cs+ solution where the triplet yield is increased by a heavy atom effect.     

A LASER FLASH PHOTOLYSIS STUDY OF PYRENE-1-ALDEHYDE. INTERSYSTEM CROSSING EFFICIENCY, PHOTOREACTIVITY AND TRIPLET STATE PROPERTIES IN VARIOUS SOLVENTS

Abstract— Triplet-and singlet-related photoprocesses of pyrene-1-aldehyde (PA) in various solvents have been investigated in detail using 337.1 and 355 nm laser flash photolysis in conjunction with time-correlated determination of fluorescence lifetimes (τ_F) and steady-state photochemical and absorption-emission spectral measurements. In benzene, the lowest triplet of PA (43 < E_T < 46 kcal/mol) has a lifetime of about 50 µs (τ_T) and displays the absorption maximum at 443 nm with a maximum extinction coefficient (ε_max) of 21000 M ^-1cm^-1; the corresponding ketyl radical has a sharp absorption maximum at 428 nm (ε_max≥ 25000 M ^-1cm^-1). The quantum yields (φ_T) of lowest triplet occupation are high in nonprotic solvents (0.6–0.8), decrease in protic solvents (alcohols) as the polarity of the latter is increased, and maintain a complementary relationship with the quantum yields (φ_F) of fluorescence. Quantum yields (φ_PC) of loss of PA due to photoreactions in some solvents have also been determined under conditions of steady irradiation at 366 nm; φ_PC is in the range 0.1–0.2 in electron-rich olefinic solvents such as cyclohexene and tetramethylethylene. These results concerning τ_F, τ_T, φ_F. φ_T and φ_PC as well as the effects of 1,2,4-trimethoxybenzene and 2,5-dimethyl-2,4-hexadiene as quenchers for fluorescence, triplet yield, and photochemistry are discussed in the light of possible state orders for PA in polar and nonpolar environments.     

MECHANISM OF PHOTOREDUCTION OF THIAZINE DYES BY EDTA STUDIED BY FLASH PHOTOLYSIS-II. pH DEPENDENCE OF ELECTRON ABSTRACTION RATE CONSTANT OF THE DYES IN THEIR TRIPLET STATE

Abstract— The pH dependence of the apparent reactivity of thiazine dyes in their triplet states has been studied in aqueous solutions, using as electron donor HY^-3, the trianionic species of ethylene diamine tetraacetic acid (EDTA), in the pH range 4–8. The pH dependence is found to be related to a change in the degree of protonation of the triplet excited dye. The apparent reactivity and lifetime of two differently protonated forms of thionine, azur B and methylene blue were determined by classical and dye-laser flash techniques, making it possible to evaluate the rate constant for electron abstraction of these molecules in their triplet states. It is found that: (a) protonation on the ring nitrogen increases the electron-abstraction rate constant of the triplet-state species about twenty-fold, and (b) methylation on the side amino groups decreases it.