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.     

KINETICS OF BACTERIAL BIOLUMINESCENCE AND THE FLUORESCENT TRANSIENT

Abstract— The addition of FMNH₂ to Vibrio harveyi luciferase at 2°C in the presence of tetradecanal results in the formation of a highly fluorescent transient species with a spectral distribution indistinguishable from that of the bioluminescence. The bioluminescence reaches maximum intensity in 1.5 s and decays in a complex manner with exponential components of 10^-1s^-1, 7 × 10^-3s^-1, and 7 × 10 ⁴s^-1. The fluorescent transient rises exponentially at 7 × 10^-2s^-3 and decays at 3 × 10^-4s^-1. The slowest bioluminescence component, comprising the bulk of the bioluminescence, decays at twice the rate of the fluorescent transient under all variations of reaction conditions: concentration of reactants, temperature 2–20°C, and aldehyde chain length—decanal, dodecanal and tetradecanal. The activation energy for both the slowest bioluminescence decay and the transient fluorescence decay is 80 kJ-mol^-1. An energy transfer scheme is proposed to explain the results where two distinct chemically energized species utilize the fluorescent transient as emitter for the slower bioluminescences, and for the faster process a fluorophore present in the protein preparation. Kinetic observations suggest that typical preparations of V. harveyi luciferase comprise 15% active protein.     

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.     

PHOTOCHEMICAL INTERACTIONS BETWEEN NITROXIDE FREE RADICALS AND PHENOTHIAZINES IN SOLUTION

Abstract— Ultraviolet irradiation of photosensitive molecules like phenothiazine derivatives leads to the formation of short lived free radicals which are able to reduce stable nitroxide free radicals generally used as spin labels. The measurement of the electron spin resonance signal decay of nitroxides offers a tool for studying the photochemical reaction of phenothiazine derivatives in solution at room temperature, in a 10^-5 to 10^-2 M concentration range. Analysis of the reaction mechanism shows that the paramagnetic nitroxide is an efficient quencher of the phenothiazine triplet state; this reaction was used to demonstrate the influence of the solvent, quenching by oxygen and the role of the chemical structure of six phenothiazine derivatives on their photoreactivity in solution.     

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.     

AN ULTRASENSITIVE BIOASSAY FOR THE DETECTION OF FUROCOUMARINS AND OTHER PHOTOSENSITIZING MOLECULES

Abstract— A photobiological assay based upon inhibition of growth in the DNA repair-deficient bacterium E. coli B _s-1, is described for the analysis of a number of photosensitizing agents. The lower limits of detection were as follows: psoralen 5 × 10^-11g; 5-methoxypsoralen 1 × 10^-9 g; 8-methoxypsoralen 1 × 10^-9 g; 4,5',8-trimethylpsoralen 1 × 10-¹¹ g; angelicin 5 × 10^-9 g; 5,7-di-methoxycoumarin 1 × 10^-7 g; isoimperatorin 5 × 10^-9 g; dictamnine 1 × 10^-8 g; oxypeucedanin 5 × 10^-7 g; 5-nitroxanthotoxin 5 × 10^-7 g; and α-terthienyl 1 × 10^-6 g. All active compounds with the exception of α-terthienyl were more easily detected by several orders of magnitude by E. coli B _s-1 than with the normal wild type E. coli. 5—Geranoxypsoralen and isopimpinellin were not active. The application of this technique, after TLC, to the analysis of complex mixtures from lemon oil, oil of bergamot, Heracleum lanatum, Angelica dawsonii , and celery and parsnip is illustrated. The bioassay described is more rapid and sensitive than previously published methods, permits replica plates to be made, and allows tentative identification of the photosensitized molecular target.     

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).     

QUENCHING OF CHLOROPHYLL FLUORESCENCE BY NITROBENZENE

Abstract—Nitrobenzene quenching of chlorophyll fluorescence in ethanol has been investigated. Steady state relative quantum yields have been measured and fluorescence decay rates were determined using both nanosecond photon counting and picosecond pulses from a mode-locked Nd³⁺ glass laser.
The fluorescence decay is described by
1( t )= I ₀ exp (- t/τ−At^1/2 )
the form predicted for decay governed by the kinetics of the continuum model of diffusion controlled reactions. From the parameters of the fluorescence decay, the encounter distance is 5–7 A° the mutual diffusion coefficient is 0.62 × 10^--⁵ cm2s^-1± 12%.
Some of the fluorescence quenching is also attributed to static quenching by a nitrobenzene-chlorophyll, ground-state complex. The equilibrium constant for formation of this ground-state complex was determined to be 4.1 M ^-1. The combined dynamic and static quenching model allows calculation of quantum yields of fluorescence in good agreement with the experimentally determined quantum yields.     

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.     

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.     

REACTIONS OF PHOTOCHEMICALLY FORMED TRANSIENTS FROM 2-NITROTOLUENE

Abstract— Kinetic data are reported for the thermal decay of colored transients formed by U.V. irradiation of aqueous solutions of 2-nitrotoluene. The transients display an acid-base equilibrium with a pK value of 3.7. The decay is catalyzed by acids and the following rate constants in liter sec^-l mole^-1 were evaluated for the base form of the transient reacting with an acid at 30.0°C: 3.5 × 10^-3 (H₂O), 2.6×10³ (CH₃COOH), 4.7×10⁴ (⁺NH₃CH₂COOH) and 4.2×10⁵(H+).     

FAR-RED INDUCED, LONG-LIVED AFTERGLOW FROM PHOTOSYNTHETIC CELLS. SIZE OF AFTERGLOW UNIT AND PATHS OF ENERGY ACCUMULATION AND DISSIPATION

Abstract— –Small amounts of N -methyl phenazonium methosulphate (PMS) added to a suspension of Chlorella pyrenoidosa accelerate the emission of the long-lived far-red induced afterglow without greatly changing the amount of light emitted. The effect is noticeable in dilute suspensions at a PMS concentration of 10^-9 M. The concept of afterglow unit is introduced and defined as that part of the sample in which the rate of energy reemission can be controlled by a single molecule of PMS. The number of chlorophyll molecules per afterglow unit is about 10⁵. It is possible that the afterglow unit is identical to the thylakoid.
The rate constant for the final first order decay phase of afterglow at room temperature is about 0.7 min^-1 without PMS and about 3 times larger for a unit with one PMS molecule.
Diuron (DCMU) lowers the rate of afterglow decay. Desaspidin on the other hand decreases the amount of light emitted without affecting the decay rate. Carbonylcyanide- m -chlorophenyl hydrazone (CCCP) decreases the afterglow over the whole time-range and increases the decay rate. A kinetic model is developed to account for the results.     

FLASH PHOTOLYSIS STUDIES OF OROTIC ACID

Abstract— The triplet state of orotic acid has been studied by flash photolysis. The rate for dimerization has been observed to vary from 2 × 10⁹ M ^-1 sec^-1 at pH 1 where both the triplet and ground state molecules are neutral, to under 10⁸ M^-1 sec^-1 above pH 9 where both the triplet and ground state molecules are doubly ionized. The p K of the triplet state has been measured as 4.6. The rate of oxygen quenching for the triplet is 2–3 × 10⁹ M^-1 sec^-1 while the rate of radiationless decay in solution is 0.73 × 10⁴ sec^-1. The triplet absorption spectra have been measured for the two ionic forms of the triplet.     

AN EPR STUDY OF THE SPECIES PRODUCED DURING THE UV PHOTOLYSIS OF HETEROCYCLIC COMPOUNDS IN METHYLTETRAHYDROFURAN AT 77 K

Abstract —The ultraviolet irradiation (290 nm ≤Λ≤ 390 nm) of indole, purine, indazole, acridine and quinoline in 2-methyltetrahydrofuran glass at 77 K produces trapped radicals. Two electron-paramagnetic-resonance (EPR) signals are found at 77 K during illumination, one at high magnetic field (3–25 times 10^-1 T) assigned to the matrix radical and the other at low field (1.3 times 10^-1 to 1–5 times 10^-1 T) attributed to the lowest triplet state of the heterocyclic molecule. Quantum yields for triplet production at 77 K are 0–34 for indole, 0.51 for purine, 0.55 for indazole, 0.15 for acridine, and 0.94 for quinoline. The rate of formation of matrix radicals varies as the n _R^th power of the incident light intensity, I ₀^nR, where 1.6 ≤ n _R=≤ 2. Solvent radical yields, which depend on the light intensity, have been determined. Under the experimental conditions, no signals attributable to trapped electrons or cations have been observed. The dependence of the reciprocal value of the rise lifetime of the low field EPR signal as a function of the intensity of exposure is in accordance with a biphotonic mechanism.     

FLUORESCENCE OF 5-METHYLCYTOSINE

Abstract— 5-Methylcytosine and 5-methyldeoxycytidylic acid are fluorescent in aqueous solution at room temperature and neutral pH. 5-Methylcytosine, 10^-3M, pH 8.5, 25°C, has a quantum yield of 5 ×10^-4, 5-Methyldeoxycitydylic acid, 10^-4M, pH 7.5, 20°C, has a quantum yield of 8 × 10^-4. Emission maxima are 2.91 and 2.80μ^-1. At pH 14, the quantum yield of 5-methylcytosine is 1.6 × 10^-2; the emission maximum is 2.82μ^-1. At pH I, the quantum yield of both compounds is less than or equal to 10^-4. Both compounds were chromatographically homogeneous, had absorption spectra which agreed with published data, and excitation spectra which agreed closely with absorption spectra.     

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.     

A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.     

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 ).     

CROCETIN, A WATER SOLUBLE CAROTENOID MONITOR FOR SINGLET MOLECULAR OXYGEN

Abstract The water soluble carotenoid crocetin has been studied as a singlet molecular oxygen monitor in D₂O solution, pD 8.4. Crocetin reacts chemically with singlet molecular oxygen with a rate constant of 4 x 10⁸ M ^-1 s^-1. The rate constant for total quenching, chemical and physical, is 2.5 x 10⁹ M ^-1 s^-1. Crocetin shows evidence for a reversible reaction with singlet molecular oxygen, as demonstrated by a fairly rapid absorption recovery after bleaching.