QUENCHING OF ENZYME GENERATED TRIPLET ACETONE BY 2-METHYL-1,4-NAPHTOQUINONE

Abstract 2-Methyl-1,4-naphtoquinone (vitamin K₃) quenches the phosphorescence from enzyme-generated triplet acetone. Concomitantly the vitamin undergoes a photochemical-like alteration as the result of transfer of the electronic energy ( k _ET– 1 ± 10⁵ M^-1). This transfer appears to be of the long-range type.     

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.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

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 TYROSINE FLUORESCENCE BY PHOSPHATE IONS: A MODEL STUDY FOR PROTEIN-NUCLEIC ACID COMPLEXES

Abstract— In order to test the ability of phosphate groups to quench the tyrosine fluorescence in nucleic acid-protein complexes, we have studied the effect of several phosphate ions on the fluorescence of tyrosine derivatives. Mono and bianions (H₂PO₄ and HPO₄^2–) which are good proton acceptors quenched the fluorescence of all the phenolic compounds studied except that of O -methyl tyrosine. With the other derivatives (tyrosine, N -acetyl tyrosinamide and lysyl-tyrosyl-α lysine) fluorescence inhibition was accompanied by the appearance of a long wavelength emission (345 nm) attributed to tyrosinate anions. The quenching of tyrosine emission was due to the deprotonation of the phenolic group promoted in the excited state by phosphate ions and leading to the weakly fluorescent tyrosinate ion. Mono and dianions of phosphate mono ester inhibited tyrosine fluorescence as did unesterified phosphates. However, phosphate diester did not have any effect on the fluorescence of tyrosine derivatives. We conclude from this study that in nucleic acid-protein complexes phosphate groups are not able to quench tyrosine fluorescence except at the end of polynucleotide chains. Since monoester and diester monoanions have a different behavior, we propose that quenching of tyrosine fluorescence by monoanions requires the formation of two hydrogen bonds. This complex cannot form with diesters which consequently do not quench tyrosine fluorescence.     

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.     

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.     

DUAL EFFECTS OF TRYPTOPHAN IN THE HORSERADISH PEROXIDASE SYSTEM THAT GENERATES TRIPLET ACETONE

When tryptophan is added to the horseradish peroxidase/hydrogen peroxide system, it markedly increases the rate of 'spontaneous' conversion of peroxidase-compound II to the native form without undergoing any alteration by spectral criteria. The D-isomer is more efficient than the L-isomer. The latter binds to the enzyme compound II, although at a site not very favourable for reduction. The accelerating effect of tryptophan is also seen when isobutyraldehyde or its enol added to the system, is converted into triplet acetone. Again the D-isomer is more efficient than the L-isomer. The D-isomer is also more efficient in quenching the acetone phosphorescence. These results indicate that, at least in the case of L-tryptophan where complexation with compound II occurs, the interaction of tryptophan with triplet acetone bound to the enzyme takes place in a ternary complex.     

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.     

FLUORESCENCE QUENCHING OF INDOLE BY DIMETHYLFORMAMIDE

Abstract— The effect of dimethylformamide on the lifetime (T) and quantum yield ( F ) of indole has been determined. Negative Stern-Volmer deviations prevail for T and F , but F ⁰/ F T⁰/T at all DMF concentrations. The deviation between F ⁰/ F and T⁰/T is ascribed to changes in F ⁰ and T⁰ induced by the change in solvent composition at the higher quencher concentrations.     

RESEARCH NOTE ON THE CHARGE-TRANSFER PATHWAY IN THE MEROCYANINE 540 TRIPLET STATE QUENCHING BY NITROXYL RADICAL

Abstract— Quenching of merocyanine 540 triplet state by nitroxyl radical has been investigated by flash photolysis. An increase of solvent polarity leads to an increase of the quenching rate. This polarity effect shows that merocyanine 540 triplet state is able to react with nitroxyl radical via a charge-transfer process. The data presented in this work are shown to be consistent with previous results obtained in micelles and liposomes.