PHOTOELECTRON QUANTUM YIELD SPECTRUM AND PHOTOELECTRON MICROSCOPY OF β-CAROTENE

Abstract—The absolute photoelectron quantum yield spectrum for β-carotene in the wavelength range 180–230 nm is reported. β-Carotene is very photoemissive over this wavelength region with photoelectron quantum yields on the order of 2 × 10^--³ electrons per incident photon at 180 nm, 4 × 10^--⁴ at 210 nm, and 3 × 10^--⁵ at 230 nm. At wavelengths longer than 240 nm, β-carotene photoemission dropped off monotonically with increasing wavelength. The photoelectron quantum yield spectrum of β-carotene is very similar to that of chlorophyll a . A photoelectron micrograph of β-carotene deposited on a thin layer of the fatty acid arachidic acid demonstrates the marked photoemission contrast between β-carotene and membrane lipid. Photoelectron micrographs of samples with β-carotene and Chl a in the same field show that prolonged (1 h) illumination in vacuo causes β-carotene to markedly fade while leaving the Chl a photoemissive. This differential bleaching of β-carotene may allow it to be distinguished from Chl in high magnification photoelectron micrographs of photosynthetic membranes.     

ON THE TRIPLET-TRIPLET ENERGY TRANSFER FROM CHLOROPHYLL TO CAROTENE IN TRITON X 100 MICELLES

Abstract— The kinetics of the triplet-triplet energy transfer of chlorophyll α (Cha) to β carotene (Car) has been investigated in Triton X100 micelles by 353 nm laser flash photolysis. This transfer consists of an intramicellar process involving pigment species located in the same micelle. A kinetic model using a bimolecular treatment leads to a rate constant of the energy transfer in the micellar phase ( k _tm≅ 6 × 10⁸ M ^-1 s^-1) lower than the previously determined values in homogeneous solvents ( k _t≅ 4.6 ≅ 10⁹ M ^-ls^-l); this result shows the high microviscosity of the micellar core. In addition, the apparent bimolecular rate constant ( k _t≅ 5.0 ≅ 10¹⁰ M ^-l s^-1) appears to be an order of magnitude higher than in homogeneous solvents. The lifetime of the carotene triplet state is the same in the hydrophobic core of Triton X100 micelles (τ a = 7.7 μs) as in organic solvents (hexane or carbon disulfide). The transfer yield is controlled by the distribution of chlorophyll and carotene molecules in the micelles.     

PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

QUANTUM YIELDS OF TRIPLET FORMATION OF SOME DERIVATIVES OF ANTHRAQUINONE

Abstract— Quantum yields of triplet formation for five amino substituted anthraquinones have been determined by the comparative actinometer method using laser flash photolysis. Substitution reduces the yields to values of ˜10^--². and the requirement of low laser intensities required high sensitivity of detection of triplet absorption. The ø_T values are compared with the quantum efficiencies of fluorescence and decomposition for the compounds, and the criteria for light stability discussed.     

ELECTRON TRANSFER FROM THE EXCITED STATE OF TYROSINE TO COMPOUNDS CONTAINING DISULFIDE LINKAGES

Abstract— The flash photolysis of aqueous solutions of tyrosine has been studied in the presence of various concentrations of the cyclic disulfide sodium lipoate (thioctic acid, Na⁺ salt). In addition to the formation of phenoxyl radicals and hydrated electrons (and possibly H atoms) from the photoionization of tyrosine, the characteristic spectrum of the radical anion RSSR^- of lipoate was also observed in neutral as well as in alkaline solutions. From the dependence of these yields upon the concentration of lipoate, it was found that a long–lived triplet excited state of tyrosine, rather than the singlet excited state, is involved in these reactions. The negative radical ions RSSR^- are formed by two distinct pathways: (a) Na⁺–lipoate reacts with the solvated electrons which are ejected from the tyrosine triplets ³Tyr → RO.+ e ^-_aq+ H⁺ followed by e ^-_aq+ RSSR → RSSR^-, and (b) by direct interaction of lipoate with triplet excited tyrosine, resulting in the transfer of a negative charge from tyrosine to the disulfide linkage. At high lipoate concentrations, the singlet excited state of lipoate is quenched, k ₄= 1.6 × 10¹⁰ M ^-1 sec^-1, but this reaction does not lead to the formation of RSSR^- radical ions.     

THE IN VITRO PHOTOCHEMISTRY OF BIOLOGICAL MOLECULES—II. THE TRIPLET STATES OF β-CAROTENE AND LYCOPENE EXCITED BY PULSE RADIOLYSIS

Abstract— –Pulse radiolysis has been used to excite the triplet states of β-carotene (τ# 9μ sec) and lycopene (τ= 8μsec) in hexane solution, both in the presence and absence of naphthalene as a triplet sensitiser. The absorption spectra of both triplets have been measured in the range 430–550 nm and have thus been extended into the region of the corresponding singlet absorptions. The overlap of the triplet and singlet spectra is discussed in relation to in vivo studies. Extinction coefficients of 1.3±0.1 × 10⁵ l/mole cm for β-carotene triplet 515 nm and 3.9±0.2 × 10⁵ l/mole cm for lycopene triplet at 525 nm were obtained. Isomerisation of the all- trans polyenes used was detected and preliminary measurements indicate that the yield of isomerisation was greater than the triplet yield. The rate of triplet energy transfer from naphthalene to β-carotene was estimated to be 1.5 × 10¹⁰ l/mole sec. The corresponding value for lycopene was 1.4× 10¹⁰ l/mole sec. The measured efficient quenching of triplet β-carotene by oxygen may occur by an energy transfer mechanism, leading to the formation of singlet oxygen (¹Δ_g. This would suggest that the triplet energy level of β-carotene lies between 121 and 94 kJ mole^-1.     

A VARIABLE TEMPERATURE, U.V. LUMINESCENCE SPECTROGRAPH FOR SMALL SAMPLES

Abstract— A variable temperature spectrometer suitable for recording phosphorescence, fluorescence and luminescence spectra is described. The instrument has been chiefly used for emission spectroscopy at temperatures between 80° and 370°K but may be used for excitation spectroscopy as well. The samples are contained in quartz tubes with inside diameters of 1.5 mm and are generally optically thick at the excitation wavelength. The sample volume can be as small as 10μl. Absolute quantum yields may be obtained with ease and the use of phase sensitive detection makes it possible to record in 30 sec a fluorescence spectrum with 30 Å resolution and a quantum yield of 10^--⁴ with a signal to noise ratio of 20. The instrument has also been useful for photochemical irradiations.     

QUENCHING OF THE FLUORESCENCE OF 8-METHOXYPSORALEN BY PROTONS

Abstract— The quenching of 8-methoxypsoralen (8-MOP) fluorescence by protons was observed to occur at the diffusion controlled rates in aqueous solutions at room temperature. Enhanced basicity of 8-MOP in the excited state compared to the ground state is expected on theoretical grounds. The fluorescence yield. which we determined as 6.3 × 10^--⁴ at pH 1 is surprisingly low and indicative of extremely fast radiationless decay pathways. The fluorescence lifetime of 8-MOP in neutral aqueous solution is on the order of 1–2 ns.     

TRIPLET FORMATION AND TRIPLET DECAY IN REACTION CENTERS FROM THE PHOTOSYNTHETIC BACTERIUM Rhodopseudomonas sphaeroides

Abstract— In the reaction center of photosynthetic bacteria, with the primary ubiquinone reduced, the triplet state P^R of the primary electron donor (a pair of bacteriochlorophylls named P) is PO ulated with a takes place in a few ns. We measured by flash absorption spectroscopy the influence of temperature on formation and decay kinetics of P^R and ³Car in the reaction center of several strains of R. sphaeroides . The rate of triplet energy transfer, measured as the decay of P^R after a flash, decreases when the temperature is lowered. Between 60 and 30 K the half-time of energy transfer becomes longer than the ³Car half-time decay (about 6 μs) and below 20 K the transfer is slower than the internal decay of P^R (about 100 μs). In several cases it is clear that P^R and ³Car decay independently and are not in thermal equilibrium. The singlet energy transfer from carotenoid to P occurs with a high efficiency at all temperatures.
The data can be accounted for on the basis of estimated energy levels of P^R and ³Car, in the context of the equilibrium ³P ←³D where ³P is the localized triplet state of P-870 and ³D is another triplet state. A reasonable kinetic scheme leads us to estimate that ³D is 0.0025 ± 0.005 eV above ³P. ³D may thus be the state observed by Shuvalov and Parson (1981). We propose that both triplet and singlet energy transfer between P and the carotenoid occur via a bacteriochlorophyll, to which the carotenoid should be tightly coupled via exchange interaction.     

INTENSITY AND TIME-DEPENDENCE OF THE CAROTENOID TRIPLET QUENCHING UNDER LIGHT FLASHES OF RECTANGULAR SHAPE IN CHLORELLA

Abstract— As is known the chlorophyll fluorescence of photosynthetizing plants is strongly quenched by carotenoid triplet states if the exciting light intensity is high enough (> 10 kW/m²). This light-induced quenching process was studied by measuring the relative yield of chlorophyll fluoresccncc excited with a pulsed argon laser at 20 C in light adapted algae as function of time (within lo μ s ) and exciting light intensity (<400 kW/m²). The experimental yield against time and yield against intensity curves have been adequately explained by a statistical model of Photosystem 2 (PS 2) units based upon the existence of freely moving excitons according to which the carotenoid triplet quenchers are randomly distributed and are perfect traps for excitons. Accepting the hypothesis that carotenoid triplet quenching occurs only in PS 2 units. it could be concluded that the height of the true zero level of PS 2 fluorescence is somewhat lower than the half value of the fluorescence level of the dark adapted state.     

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.     

CAROTENOID TRIPLET STATE AND CHLOROPHYLL FLUORESCENCE QUENCHING IN CHLOROPLASTS AND SUBCHLOROPLAST PARTICLES

Abstract— Absorption changes attributed to the triplet state of carotenoids and to primary electron donors (P-700. P-680)^: and fluorescence quenching at several wavelengths have been measured with a single apparatus. following flash excitation with a dye or a ruby laser. Spinach chloroplasts as well as subchloroplast particles enriched in Photosystem-1 (F₁), Photosystem-2 (F₁) or the light-harvesting Chl a/h (F_III) have been examined at temperatures varying between 5 and 294 K.
The triplet state of carotenoids has been identified on the basis of its difference spectrum (having a peak at 515 nm) and decay kinetics (⋍ 7 µs at low temperature; accelerated by O₂ at 294 K). It is formed in all of the materials studied. The quantum yield of carotenoid triplet formation in chloroplasts increases at low temperature, but less than the fluorescence yield.
In most cases the fluorescence quenching recovers approximately with the same kinetics as the decay of the carotenoid triplets. The fluorescence recovery is, however, significantly faster for chloroplasts at 730 nm. Fluorescence quenching occurs in all types of materials. The ratio of fluorescence quenching to the concentration of carotenoid triplets varies with the material, being maximum in chloroplasts and minimum in F_m particles.
We conclude that the formation of the carotenoid triplet state is not limited to a few sites in the chloroplast and that a carotenoid triplet is a quencher of chlorophyll fluorescence. A detailed comparison of carotenoid triplets and fluorescence quenching gives some information concerning the organization of the pigments in the photosynthetic apparatus.     

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.     

PHOTOSENSITIZED CYCLODIMERIZATION OF 5-FLUOROURACIL

Abstract—The photosensitization by acetone or N-methyllutidone yields a C₄-cyclodimer from 5-fluorouracil in aqueous solution. with a quantum yield of 2 × 10^--³. The dimer isolated has been characterized by UV, IR. NMR, and mass spectra. The stereochemistry of the dimer has been determined to be anti head-to-tail configuration from the NMR spectral analysis.     

SPINACH FERREDOXIN REDUCTION BY MEMBRANE-BOUND CHLOROPHYLL TRIPLET STATE VIA A DIQUAT MEDIATOR

Abstract Laser flash photolysis experiments have shown that the diquat analog containing a propylene bridge (PDQ²⁺), when electrostatically bound to negatively-charged vesicles containing chlorophyll, is able to mediate the rapid reduction ( k = 1.1 × 10⁵ s^-1) of spinach ferredoxin via electron transfer quenching of triplet state chlorophyll. The kinetics of formation and decay of reduced ferredoxin are consistent with a mechanism involving complex formation between oxidized ferredoxin and vesicle-bound PDQ²⁺. Under optimal conditions, approximately 15% of the quenched triplets yield reduced ferredoxin. This process is a model for soluble ferredoxin reduction which occurs in green plant photosystem I, and results in an appreciable storage of electromagnetic energy in the reaction products.     

AN INVESTIGATION OF RETINAL AS A SOURCE OF SINGLET OXYGEN

Abstract— All -trans retinal is dissolved in alcohols and illuminated at 365 nm in the presence of a singlet oxygen acceptor, 2,5-dimethylfuran. Illumination produces the photosensitized oxidation of the acceptor which is measured by the disappearance of its 215 nm absorption band. A kinetic study is carried out and β_DMF is 1.6 × 10^--⁴ M . The quantum yield of ¹O₂ production from the light-excited retinal is estimated to 0.096. The retinal sensitized photooxidation of dimethylfuran is inhibited by a ¹O₂ quencher, 1,4-diazabicyclo(2,2,2)-octane, and enhanced by deuteration of the solvent. Deuterated solvents are known to increase ¹O₂ lifetime.
The production of ¹O₂ from retinal is briefly discussed in relation to the damage which may be induced by light in the visual cells.     

EFFECT OF TEMPERATURE AND pH ON THE QUENCHING OF TRIPLET LUMIFLAVIN IN THE PRESENCE AND ABSENCE OF FERRI- AND FERROCYANIDE

Abstract— –Flash photolysis at 450 nm over the temperature range 0.8–60°C was used to determine Arrhenius parameters for the first and second order disappearance of triplet lumiflavin (1.66 µ .M ) at a flash energy of 2 kj in deaerated phosphate buffer at varying pH:
³Lf → Lf₀
³Lf +³Lf → Lf₀+ Lf₀
Arrhenius parameters were also determined for the pseudo first-order quenching of triplet lumiflavin by 10 µ M ferri- and ferrocyanide ions,
³Lf + Fe³⁺→ Fe³⁺→ Lf₀+ Fe³⁺ (energy transfer)
³Lf + Fe²⁺→ Lf^-+ Fe³⁺ (electron transfer)
and for disappearance of the semireduced lumiflavin in the presence of ferrocyanide at pH 6.8, by the second-order reaction
Lf^-+Lf ^-→ Lf₀+ Lf⁼.     

LASER PHOTOLYSIS STUDIES OF QUINONE REDUCTION BY PHEOPHYTIN a IN ALCOHOL SOLUTION

Abstract- Upon laser photolysis of pheophytin-benzoquinone solutions in ethanol, transients due to the pheophytin triplet state (P_t), an exciplex (Pδ⁺ Qδ^-), the pheophytin cation radical (P⁺) and the semiquinone radical (Q^-) can be observed. Kinetic analysis indicates that the evolution of these transients at times longer than one microsecond is due to the decay of the exciplex with the concomitant formation of P⁺ and Q^-, reverse electron transfer to form P and Q, solvent oxidation by P⁺, and Q^- disproportionation. In support of the suggested solvent oxidation reaction, a large deuterium isotope effect is observed upon changing the solvent from methanol to its fully-deuterated counterpart. Comparisons are made between these results and those obtained with chlorophyll as described in the preceding paper.     

Transitory forms of carotenoids: triplet state and radical cation

Abstract— Reactions between toluidine blue (a thiazine dye) and carotenoids (β-carotene and zeaxanthine) were studied by flash photolysis, in ethanolic solutions. Two types of reactions were evidenced:

• 1 a triplet-triplet energy transfer from the triplet state of monoprotonated toluidine blue to the carotenoid whose triplet state decays rapidly (t_1/2= 5μsec).
• 2 an electron transfer from the carotenoid to the mono- and bi-protonated triplet states of toluidine blue. This produces a radical-cation of the carotenoid (Car⁺) which decays slowly (t_1/2? 200 μsec) and has a strong absorption band around 900 nm.

     

FORMATION AND CHEMILUMINESCENT DECOMPOSITION OF DIOXETANES IN THE GAS PHASE

Abstract— High resolution chemiluminescence spectra have been obtained of the singlet electronically excited products of O₂(¹Δ) plus alkene, dioxetane forming, reactions. The experiments were conducted in a flow apparatus at pressures of 1–5 torr. The spectra are a measure of the unrelaxed initial distribution of energy in the excited product. Results are reported for ethylene, 1, 1-difluoroethylene. methyl vinyl ether, ethyl vinyl ether, n -butyl vinyl ether, ketene, ketene-d₂, allene, unsymdimethyl allene, dimethyl ketene, 2-methoxy propene, 1-ethoxy propene, 2-bromo propene, and N, N- dimethyl isobutenyl amine. Chemiluminescence activation energies, representing the cycloaddition process, and absolute quantum yields for singlet excited product, ranging from 10^--⁴ to 2.5 × 10^--². are reported for 10 alkenes. Several of the reactions, 1,1-difluoroethylene, ketene, ethylene and allene give formaldehyde ¹ nπ* product with excess vibrational-rotational energy and a higher quantum yield than reactions not displaying this phenomenon. This is an indication of at least partially statistical partitioning of the energy in excess of that needed to electronically excite the formaldehyde. The experiments with ketene and ketene-d₂ provide the first evidence for the existence of unsubstituted 1,2-dioxetanone. The results from several of the experiments, particularly those with 2-methoxy propene and I-ethoxy propene are consistent with the mechanism of Goddard, which predicts regioselective and stereoselective attack of O₂(¹Δ) upon alkoxy substituted alkenes having allylic hydrogen.