LONG-RANGE TRIPLET-SINGLET ENERGY TRANSFER FROM ENZYME GENERATED TRIPLET ACETONE TO XANTHENE DYES

Enzyme-generated and protected acetone transfers its energy to xanthene dyes by a nontrivial process. The relative populations of the S₁ state are indicative of the operation of a heavy atom effect, being 1:15:100 for fluorescein, eosine and Rose Bengal. On the other hand Stern-Volmer constants and k _ET⁰ values are similar for the three dyes and are remarkably higher than for collisional processes.
It is tentatively suggested that at least for eosine and Rose Bengal, a triplet-triplet cxciton transfer may occur to populate an upper triplet state of the dye followed by heavy-atom enhanced intersystem crossing to the S₁ state.     

QUENCHING OF CHEMICALLY and ENZYMICALLY-GENERATED TRIPLET ACETONE BY TYROSINE and 3,5-DIHALOGENODERIVATIVES

Tyrosine and especially its 3,5-dihalogenoderivatives quench acetone triplets. When the excited acetone is generated free in solution, the Stern-Volmer plots for the quenching by these species, monitored via the sensitized emission of the 9,10-dibromoanthracene-2-sulfonate ion, are linear. When triplet acetone is generated enzymically by the peroxidase-catalyzed aerobic oxidation of isobutyral-dehyde, the Stern-Volmer plots for the quenching of the acetone phosphorescence curve upwards in the case of 3,5-dibromotyrosine and even more markedly with 3,5-diiodotyrosine. Quenching appears likely to occur by triplet-triplet energy transfer and especially in the case of the phenoxide form, also by electron transfer. The curvature denotes a static contribution to quenching favoured by the enzyme.     

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.     

QUENCHING OF A RETINAL TRIPLET STATE BY A NITROXYL RADICAL

Abstract— The long lived triplets from all-trans retinal and 11-cis retinal are quenched by a nitroxyl radical, 4-hydroxy-2,2,6,6-tetramethylpiperidinoyl, with essentially identical rate constants. The rates vary with solvent but do not correlate with solvent polarity. The results confirm implications of earlier work with oxygen quenching and are compatible with the view that isomerization occurs in non-relaxed triplets or that the triplet (or triplets) observed spectroscopically decay by way of a single triplet state which has a small electronic energy gap to ground state isomers.     

PEROXIDASE CATALYZED GENERATION OF TRIPLET ACETONE

Abstract— Triplet acetone generated in the isobutanal/horseradish peroxidase/O, system is quenched by collisional agents of the diene type. A Stern-Volmer analysis indicates that collision is considerably impeded, the barrier being largely entropic. Triplet-triplet energy transfer to biacetyl is also observed. Quenching of triplet acetone by energy transfer to 9 ,10-dibromoanthracene-2-sulfonate (DBAS) is a much faster process than energy transfer to these collisional agents, in accordance with the earlier inference that transfer to DBAS is a long-range triplet-singlet transfer process. The fraction of the triplet acetone which undergoes reduction to isopropanol is not interceptable by quenchers.
Absorption and circular dichroism studies suggest that peroxidase shuttles between Compound II and I when generating the precursor hydroperoxide.     

PHOTOCHEMICAL-LIKE BEHAVIOUR OF RIBOFLAVIN IN THE DARK PROMOTED BY ENZYME-GENERATED TRIPLET ACETONE

Abstract— Triplet acetone obtained enzymically by the isobutyraldehyde/peroxidase/O₂ system transfers its energy to the riboflavin, which changes into a decomposition product of lumichrome type and produces aggregates. When riboflavin and tryptophan are added to this system, further products—formylkynurenine and an adduct between the riboflavin and the tryptophan—are formed. The adduct can be separated by gel filtration and ion exchange chromatography and is similar to that prepared by irradiating riboflavin in the presence of tryptophan.     

RED EMISSION FROM CHLOROPLASTS ELICITED BY ENZYME-GENERATED TRIPLET ACETONE AND TRIPLET INDOLE-3-ALDEHYDE

—Enzyme-generated triplet acetone and triplet indole-3-aldehyde transfer energy very efficiently to chloroplasts, as indicated by the intensity of the sensitized red emission that is observed. The intermediacy of excited species of oxygen (¹O₂, O₂⁻, HO) has been excluded. Our results open the way for investigating energy transfer in architecturally organized systems in the absence of light.     

ESR STUDY OF ORIENTED TRIPLET MOLECULES GENERATED BY EXCITATION WITH POLARIZED LIGHT

Abstract— The lowest-lying triplet states of a variety of aromatic molecules and complexes have been generated by the irradiation of these compounds in solvent glasses with plane-polarized light. Measurements of the allowed (Δ M=± 1) ESR transitions clearly demonstrate that the triplets so formed are oriented with respect to the external magnetic field. By this method the triplet zero-field splitting parameters, D and E , can be evaluated simply and reliably. Intramolecular energy transfer is postulated to explain the triplet spectra of Zn( o -phen)₂(NO₃)₂ and Zn( o -phen)₃(NO₃)₂. It was observed that in triplet-triplet energy transfer from benzophenone to naphthalene there is no apparent orientation requirement between the donor and acceptor molecules. Further areas of significance and application of this technique are suggested.     

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).     

POSSIBLE IN VIVO FORMATION OF LUMICHOLCHICINES FROM COLCHICINE BY ENDOGENOUSLY GENERATED TRIPLET SPECIES

Abstract In Colchicum autumnale L., colchicine is accompanied by its photoproducts, mainly β-lumicholchicine, even in parts not exposed to light such as the corm of plants grown in the dark. The transformation of colchicine into lumicolchicines can be efficiently accomplished in vitro by energy transfer from enzyme-generated triplet species to colchicine, thus providing additional evidence for'photobiochemistry without light'.     

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).     

CONJUGATED DIENE FORMATION PROMOTED BY TRIPLET ACETONE ACTING UPON ARACHIDONIC ACID

Abstract— Post UV-B(280–320 nm) exposure to UV-A(320–400 nm) reverses pyrimidine dimers in the epidermal DNA of the South American opossum Monodelphis domestica [Ley, R. D. (1984) photorepair of pyrimidine dimers in the epidermis of the marsupial Monodelphis domestica. Photochem. Photobiol . 40 ,141–143.] To demonstrate that the observed photorepair is mediated by an enzyme, we have isolated a DNA photolyase from the opossum. DNA photolyase from liver was purified 3000-fold by ammonium sulfate fractionation and phenylsepharose, hydroxylapatite, DEAE-cellulose and DNA-cellulose column chromatography. Heat denaturation (60°C for 4 min) completely eliminated the photoreactivating activity. The enzyme was active in the pH range of 5.5 to 8.5 with a pH optimum of 7.5. The enzyme has an apparent molecular weight of 32 000 under nondenaturing conditions. The activity of the enzyme was not affected by sodium chloride up to 250 m M . The action spectrum for the purified DNA photolyase showed activity in the range of325–475 nm with peak actvity at 375 nm.     

A METHOD OF TREATING PHOTOCHEMICAL TRIPLET QUENCHING DATA

Abstract— A modified Stern-Volmer plot method is proposed for treatment of quantum yield data where both reactive photoexcited singlet and triplet states are present in solution. The method may be applied when the reactions occurring are zeroth order in ground state photochemical substrate and allows the separation of singlet and triplet state contributions to the total quantum yield and the calculation of kinetic lifetimes without going to very high quencher concentrations. The method is illustrated through its application to literature data on the Type II photoelelimination reaction of 2-hexanone.     

QUENCHING OF THE TRIPLET STATE OF CHLOROPHYLL a BY ASCORBIC ACID AND ASCORBATE IN ETHANOLIC SOLUTION

Abstract— Ascorbic acid and ascorbate in chlorophyll ethanol solution were found to be fairly efficient quenchers of the chlorophyll triplet state; comparable to the efficiency of ascorbic acid as a quencher in aqueous pyridine solution.
It has been well established that ascorbic acid quenches the triplet state of chlorophyll in aqueous pyridine solution.^(1,2) The bimolecular quenching constant, kQ , is very much less than that for O₂ or quinine.^(3,4)
Information regarding the quenching of the triplet state of chlorophyll by ascorbic acid in ethanolic solution is lacking. There has been some question as to whether ascorbic acid reduces photoexcited chloro-phyll-ethanolic solution because of its high oxidation potential, or because like the ascorbate ion, it acts only as a quencher; both ascorbic acid and ascorbate in high concentrations gave low quantum yields.⁽⁵⁾ The quenching of the triplet state by ascorbic acid and ascorbate was determined by the flash-photolytic method.     

QUENCHING OF TRIPLET BENZOPHENONE BY VITAMINS E and C and BY SULFUR CONTAINING AMINO ACIDS and PEPTIDES

Abstract— The quenching rate of triplet benzophenone in water and/or water mixtures has been determined employing vitamin C, vitamin E, cystine, cysteine, reduced and oxidized glutathione, methionine and DL-penicillamine. In these systems, the ketyl radical quantum yield and the benzophenone photoreduction yield have also been measured. The ketyl quantum yield is 1.0 in presence of vitamin C and smaller than 0.3 in presence of glutathione, cysteine and cystine. The data imply that quenching by thiols and disulfides takes place, at least in very polar solvents, mainly by a mechanism involving charge transfer intermediates.     

CRYSTAL STRUCTURE OF 2-METHYL-3-PHENYLACRYLOYL-QUINOXALINE 1,4-DIOXIDE

<正> C18H14N2O3,Mr=306. 32,Monoclinic,P21/b,a = 5. 579(1),b = 8. 963 (2),c = 29. 405(7) A,γ= 92. 62(2)°,V= 1468. 8(7) A3.Z=4, Dx= 1.385 gcm-3, CuKa radiation, μ= 7. 45 cm-1,F(000) = 640,final R= 0. 039,Rw= 0. 045 for 1006 observed (I>3σ(I)) reflections. The quinoxaline part of the molecule is virtually planar. The dihedral angle between the benzene ring of the phenylacryloyl and quinoxaline ring is 99. 6°.     

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.     

PHOTO-OXIDATION OF THYMINE SENSITIZED BY 2-METHYL-1,4-NAPHTHOQUINONE: ANALYSIS OF PRODUCTS INCLUDING THREE NOVEL PHOTO-DIMERS

Abstract —The near-UV photosensitization of thymine by 2-methyl-1,4-naphthoquinone in oxygenated aqueous solution leads to the formation of nine major thymine-derived products. Six of the photoproducts, representing 70% of the initial thymine loss, are also known to be formed by γ-ray or X-ray irradiation of oxygenated water solutions of thymine. They include the cis and trans isomers of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine, the cis and trans thymine glycols, 5-hydroxy-5-methylhydantoin, and N-formyl-N'-pyruvylurea. The three remaining major products, accounting for a further 25% of the initial thymine loss, are thymine di-adducts of a type not previously reported. Preliminary characterization of these novel photoproducts, based on their UV absorption, mass spectra, and proton and carbon-13 nuclear magnetic resonance spectra, suggests that in each of the three, the thymine moieties are linked by an N(1)-C(5) or N(1)-C(6) bond. A thymine cation radical is implicated as the common precursor of all 9 products. Accordingly. pyrimidine photosensitization by 2-methyl-1,4-naphthoquinone serves as a model system for studying the chemical consequences of radiation-induced ionization of the DNA bases.     

ENERGY TRANSFER FROM ENZYME-GENERATED TRIPLET ACETONE TO RIBOFLAVIN PERTURBED BY MOLECULES RELATED TO THYROXINE

Abstract— The rate of energy transfer from enzyme-generated triplet acetone to riboflavin increases by three orders of magnitude when the flavin is charge-transfer complexed with 3 ,5-dihalogenotyrosines or with thyroxine. A very fast long-range energy transfer to form the first excited singlet state of riboflavin in the complex, followed by a very efficient intersystem crossing to the triplet manifold, is postulated to account for the results.