DELAYED-LIGHT STUDIES ON PHOTOSYNTHETIC ENERGY CONVERSION-IV. EFFECT OF TRIS POISONING, AND OF ITS REVERSAL BY HYDROGEN DONORS, ON THE MILLISECOND EMISSION FROM CHLOROPLASTS*

Abstract— Destruction of the oxygen-evolving activity of chloroplasts by treatment with 0.8 M Tris-HC1 results in an extremely rapid dark decay of millisecond delayed light. Addition of electron acceptors such K₃Fe(CN)₆ or NADP⁺ does not change the decay characteristics of this msec delayed light. Artificial electron donors such as DPC partially restore the msec delayed emission to the slowly decaying situation which is found in control chloroplasts. Addition of electron acceptors to this photochemically competent system results in more rapid decay and in an increase of emission at 1 msec, as in control chloroplasts. We suggest on the basis of the delayed light data that Tris treatment induces a rapid side reaction which uselessly dissipates the oxidizing and reducing power which is stored by Photoreaction II. Artificial electron donors allow the Tris-poisoned photoreaction to store energy long enough for utilization of the energy by normal photosynthetic reactions, as shown by the flattening of the delayed light curves. In the restored system the normal reactions of electron transport are thus able to compete with the Tris-induced side reaction. This interpretation is supported by the finding that the restored system requires higher exciting intensities for saturation of NADP⁺ reduction than the control system.     

THE EFFECT OF INHIBITORS AND UNCOUPLERS OF PHOTOSYNTHETIC PHOSPHORYLATION ON THE DELAYED LIGHT EMISSION OF CHLOROPLASTS*

Abstract— Measurements were made of the 3.7 msec delayed light emission of chloroplasts treated with a variety of agents which affect the rate of electron transport (Hill reaction) or photosynthetic phosphorylation. The presence of the electron acceptors ferricyanide or pyocyanine increased delayed light emission. Inhibitors of electron transport (3-(3,4-dichlorophenyl)-1, -1-dimethylurea or 1,10(ortho)-penanthroline) inhibited delayed light emission. The addition of a phosphate acceptor system inhibited delayed light emission. This inhibition was reversed by inhibitors of the phosphorylation reaction, e.g. Dio-9 or phlorizin. From these results it was concluded that the 3.7 msec delayed light emission probably occurs as a result of back reactions of intermediates in the coupled electron transport and photosynthetic phosphorylation systems.     

Visible light induced ocular delayed bioluminescence as a possible origin of negative afterimage

The delayed luminescence of biological tissues is an ultraweak reemission of absorbed photons after exposure to external monochromatic or white light illumination. Recently, Wang, Bókkon, Dai and Antal (2011) [10] presented the first experimental proof of the existence of spontaneous ultraweak biophoton emission and visible light induced delayed ultraweak photon emission from in vitro freshly isolated rat's whole eye, lens, vitreous humor and retina. Here, we suggest that the photobiophysical source of negative afterimage can also occur within the eye by delayed bioluminescent photons. In other words, when we stare at a colored (or white) image for few seconds, external photons can induce excited electronic states within different parts of the eye that is followed by a delayed reemission of absorbed photons for several seconds. Finally, these reemitted photons can be absorbed by non-bleached photoreceptors that produce a negative afterimage. Although this suggests the photobiophysical source of negative afterimages is related retinal mechanisms, cortical neurons have also essential contribution in the interpretation and modulation of negative afterimages.     

Fragmentation of (LiF)(n)Li(+) clusters in the acceleration region of TOF spectrometers

The binary decay of ionized clusters in the extraction region of time-of-flight (TOF) spectrometers is analyzed. The dynamics of the fragments is studied and an analytical expression for the TOF peak shape is deduced; simulations are performed for linear spectrometers of different configurations. The questions addressed refer to the design of TOF spectrometers to improve their accuracy in the determination of metastable-state mean lives, the identification of precursor masses and the investigation of desorption mechanisms. As an illustration, the method is applied to the decay of positive ion clusters (LiF)(n)Li(+) for both spontaneous and collision-induced fragmentation processes. No clear evidence of delayed emission is found. The bumps observed in the TOF spectrum are due to tertiary ions emitted by the LiF target sputtered by secondary ions produced in the grid, a process that increases with higher target bias. The main cluster fragmentation observed is (LiF)(3)Li(+*) decaying preferentially into (LiF)Li(+); the data are compatible with a spontaneous decay of metastable clusters with mean lives of 20-30 ns.     

ROOM TEMPERATURE TIME-RESOLVED IN μs RANGE DELAYED LUMINESCENCE OF CHLOROPHYLL a AND CHLOROPHYLL-LUTEIN MIXTURE SOLUTIONS

Using time-resolved in μS range luminescence spectroscopy, we observed at 20°C the emission of chlorophyll a, pheophytin a and chlorophyll a-lutein mixture solutions. This delayed emission exhibits several maxima in the650–750 nm region. The positions and kinetics of decay of delayed emission bands depend on chlorophyll concentration, and vary as a result of pheophytinization and addition of lutein. Our results can be explained by supposition that upon excitation, charge transfer species are formed in various pigment complexes. The back electron transfer reactions yield chlorophyll excited singlet states contributing to observed delayed emission. Delay in emission seems to be due also to the trapping of excitation on the triplet states of various forms of pigment and its detrapping with the participation of thermal energy followed by energy transfer to the forms of pigment characterized by different decay times.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

RESOLUTION OF TWO EMISSION SPECTRA FOR TRYPTOPHAN USING FREQUENCY-DOMAIN PHASE-MODULATION SPECTRA

We describe a novel application of frequency-domain fluorometry which allows resolution of the decay times and emission spectra of samples which display multi-exponential decay kinetics. This method does not require any previous knowledge about the decay times or any assumptions about the shape of the emission spectra. We record the wavelength-dependent phase angles and modulations (phase angle and modulation spectra) using a number of light modulation frequencies. The data is analyzed by non-linear least-squares to recover the emission spectra and their associated decay times. Phase and modulation spectra (PM Spec) were used to recover the emission spectra associated with the two decay times of tryptophan at pH = 7 (0.54 and 3.44 ns). The emission spectra of these components are centered at 340 and 355 nm, respectively, with the amplitude of the 0.54 ns component contributing 6% to the total emission. These results are in agreement with previous time-resolved studies by Szabo and Rayner [J. Am. Chem. Soc. 102, 554-563 (1980)]. Control experiments were performed on mixtures of N-acetyl-L-tryptophanamide (NATA) and PPD, which demonstrate our ability to recover the spectra and decay times from two component mixtures. NATA itself displayed a single decay time and only one emission spectrum.     

Rate and driving force for protonation of aryl radical anions in ethanol

Aryl radical anions created in liquid alcohols decay on the microsecond time scale by transfer of protons from the solvent. This paper reports a 4.5 decade range of rate constants for proton transfer from a single weak acid, ethanol, to a series of unsubstituted aryl radical anions, Ar-*. The rate constants correlate with free energy change, DeltaG(o), despite wide variations in the two factors that contribute to DeltaG(o): (a) the reduction potentials of the aryls and (b) the Ar-H* bond strengths in the product radicals. For aryl radical anions containing CH2OH substituents, such as 2,2'-biphenyldimethanol*- which is protonated with a rate constant of 3x10(9) s(-1), the faster rates do not fit well in the free energy correlation, suggesting a change in mechanism.     

ACTIVATION OF LUCIGENIN CHEMILUMINESCENCE BY SERRATIA MARCESCENS

Abstract Cell–free extracts and intact cells of Serratia marcescens were found to activate lucigenin (10,10 -dimethy1-9,9-biacridylium nitrate) chemiluminescence in the absence of either added H²O² or alkali. Light emission proceeded in alcoholic solvents and, in general, the intensity decreased with increasing length of the alcoholic carbon chain. Th e intensity of bacterially activated lucigenin chemiluminescence increased in a logarithmic linear manner with increasing methanol concentrations, maximum intensities occurring with 90% methanol. Other organic lucigenin solvents also supported the bacterially activated light emission process, although not to the same extent as 90% methanol. The addition of KOH to methanol failed to enhance chemiluminescence. The luminescent process was charaterized by the attainment of peak light emission three seconds after the initiation of the reaction, followed by rapid decay to a low constant light level. The bacterial activation of lucigening chemiluminescence was found to be enhanced by the inclusion by the inclusion of fluorescein in the neutral methanol solvent.     

ELECTROCHEMICAL GENERATION OF SOLUTION LUMINESCENCE—III.: EFFECT OF OXYGEN ON QUANTUM YIELD AND KINETICS OF LUMINOL

Abstract -The growth and decay of light emission were examined for luminol as a funtion of oxygen concentration in aqueous alkaline solutions during and after the application of a controlled potential, square-wave electrochemical pulse. The results indicate that, in the rise portion of the light, the rate of electrochemical oxidation of luminol governs the light emission rate; while in the decay portion, the rate is first order and is independent of oxygen concentration. Quantum yields based on integration of the total light emitted also appear to be independent of oxygen above a threshold value. These results are consistent with evidence that the emitting state is an excited singlet.     

Ultraweak and Induced Photon Emission After Wounding of Plants

The ultraweak and induced photon emission were measured by a single photon counting equipment (Photomultiplier Hamamatsu R562) on Cucurbita pepo variaca styriacae after wounding. Wounding significantly changes the emission from a stationary to a nonstationary state and the shape of the decay curve obtained after light illumination. The rise in the ultraweak photon emission depends on the kind of wounding and its localization on the plant. The decay curves obtained after wounding could be better fit by an exponential function than by a hyperbolic one. So the biophoton emission correlates with physiological and bioelectrical changes like membrane depolarizations as they also depend on the kind of injury.     

Urea-based ruthenium(II)-polypyridyl complex as an optical sensor for anions: synthesis, characterization, and binding studies

A new ruthenium(II) complex [Ru(bpy)2(1-(6-nitro-[1,10]phenanthrolin-5-yl)-3-(4-nitrophenyl)-urea)] (bpy=2,2'-bipyridyl) was synthesized and characterized using standard analytical and spectroscopic techniques. Detailed absorption, emission, and 1H NMR spectral studies revealed that this receptor molecule acts as a sensor for F-, CH3COO-, and H2PO4- in acetonitrile solution. Binding of these anions caused an appreciable change in the color of the acetonitrile solution, which could be detected with the naked eye. At relatively lower concentration of anions, 1:1 H-bonded adduct was formed; however, at higher concentration, classical Br?nsted acid-base-type reaction prevailed. The relative binding affinity of different anions toward this receptor was evaluated and was rationalized with quantum chemical calculations. Narrowing of the gap between the highest occupied molecular orbital and lowest unoccupied molecular orbital energy levels on deprotonation of the receptor molecule caused a faster decay of the luminescence lifetime for the Rudpi-->Lpi*/bpypi*-based triplet excited state.     

Electron transfer at the single-molecule level in a triphenylamine-perylene imide molecule.

Photoinduced electron transfer (ET) processes in a donor-acceptor system based on triphenylamine and perylene imide have been studied at the single-molecule (SM) and ensemble levels. The system exists as two isomers, one of which undergoes forward and reverse ET in toluene with decay constants of 3.0 and 2.2x10(9) s(-1), respectively, resulting in the dual emission of quenched and delayed fluorescence while the other isomer remains ET-inactive. The fluorescence of both isomers is heavily quenched in the more polar solvent, diethyl ether, by ET. A broad range of ET dynamics is seen at the SM level in polystryene with the two isomers nonresolvable indicating that the local nanoenvironment of the SMs varies considerably throughout the polymer matrix. Both the electronic coupling and the driving force for ET are shown to influence the ET dynamics. Many fluorescence trajectories of SMs show long periods (tens of milliseconds to seconds) where the count rate is attenuated either partly (a "dim" state) or to the background level (an "off-time"). During these periods, the reduction or interruption of emission is attributed to cycles of rapid charge separation followed by charge recombination to the ground state reducing the fluorescence quantum yield of the SM.     

ANALYSIS OF MICROSECOND FLUORESCENCE YIELD AND DELAYED LIGHT EMISSION CHANGES AFTER A SINGLE FLASH IN PEA CHLOROPLASTS: EFFECTS OF MONO- AND DIVALENT CATIONS

Abstract. New results are presented on the effects of mono- and divalent cations on concurrent changes in the microsecond yields and kinetics of chlorophyll a fluorescence and delayed light emission, and the light saturation curve for the latter at 100 μs, following a 10 ns flash at 337 nm. (1) The fluorescence yield increases exponentially from 3 to 30 μs (lifetime, τ, 6.4 ± 0.6/μs), and decays biphasically between 50 and 800μs. (2) The delayed light emission decays biphasically with two exponential phases: fast phase, T= 7–10μs, and slow phase, T= 33–40μs. (3) The light saturation curve for 100μs delayed light emission is satisfactorily represented by a one-hit Poisson saturation curve. (4) Addition of 5 mM NaCl to salt-depleted chloroplasts decreases (by as much as 40%) the yields of μs fluorescence and delayed light emission, and the subsequent addition of 5mM MgCl₂ increases the yields (≤2 × over samples with only NaCl). (5) The fluorescence yield rise and delayed light emission decay kinetics are independent of low concentrations of cations. The lifetime of the fast phase of fluorescence decay changes from ?90μs to ?160μs, when Na⁺ or Na⁺+ Mg²⁺ are added. Based on a detailed analysis presented in this paper, the following conclusions regarding the effects of low concentrations (few mM) of mono-and divalent cations in sucrose-washed chloroplasts at room temperature are made: (a) Na⁺ decreases (?6%) and Mg²⁺ increases (? 20% compared with the Na⁺ sample) the sensitization of photosystem II photochemistry: this effect is small, but significant. (b) Na⁺ increases and Mg²⁺ decreases the efficiency for radiationless transitions in singlet excited Chl a in the antenna and closed reaction center of PS II; this includes non-radiative energy transfer to PS I, intramolecular intersystem crossing and internal conversion. The ratio of the sum of the rate constants for radiationless transitions to that for fluorescence increases by ? 2-fold upon the addition of Na⁺, and is completely reversed by the addition of Mg²⁺. (c) The rate constant for the re-oxidation of Q^- decreases (about 50%) in the presence of Na⁺ or Na⁺+ Mg²⁺. These conclusions imply that cations produce multiple changes in the primary photoprocesses of PS II at physiological temperatures. It is proposed that these changes are mutually independent and can co-exist.     

The effect of dose on light-sensitivity of radicals in alanine EPR dosimeters

In this work we present a study of light-induced effects on free radicals and their transformations in gamma-irradiated pure L-alanine and in commercially available alanine detectors: rods, pellets and films. Samples irradiated to doses from 2 Gy to 4000 kGy were exposed to light from a fluorescent lamp and to ordinary daylight. The observed changes in EPR spectra of the samples were analyzed with regard to their intensity and shape. The shape analysis was based on numerical decomposition of the measured spectra into model spectra reflecting contributions of R1, R2 and R3 radical populations in the samples. The illumination of alanine dosimeters resulted in significant decrease of the central EPR line and was accompanied by distinct variations in the shape of EPR spectra. The rate of light-induced decay in spectra amplitude was found to be dependent on dose of ionizing radiation--the sensitivity to light was decreasing with increase in dose in all detectors in the 2-5x10(5) Gy dose range. The exposure of gamma-irradiated (to 300 Gy) alanine to normal, diffused daylight resulted in decay of the signal amplitude at rate about 0.5% per week. It was shown that decay in the R1 component was responsible for the observed reduction of the spectra amplitude. The observed increase in R2 contributions in samples exposed to light confirmed a hypothesis of R-->R2 radical transformations promoted by visible light. The reported effects indicate a necessity of protection of irradiated dosimeters from their prolonged exposure to light.     

INDUCTION KINETICS OF DELAYED LUMINESCENCE IN PHOTOSYNTHETIC ORGANISMS AS MEASURED BY AN LED-BASED PHOSPHORIMETER

Abstract— Induction kinetics of delayed luminescence of photosynthetic organisms were measured with a computer-controlled spectrophotometer based on a 3 × 3 array of novel, red superbright light-emitting diodes (LED). Compared to the conventional Becquerel phosphoroscope, the computer-controlled setup is characterized by superior measuring properties, simplicity, small size and a rugged construction with no mechanical moving parts. Time resolution of approximately 10 ms allows the observation of early processes of photosynthesis reflecting the light-induced build-up of an electrochemical potential across thylakoid membranes. A complex kinetic time course of light induction of delayed luminescence is observed in all dark-adapted photosynthetic organisms, but the crude pattern is always similar: a fast initial increase within 40 ms, followed by a slower drop within some 300 ms, a second increase to a maximum within a few seconds and finally a slow decrease within 30 s toward a low emission steady state. The results are interpreted in terms of photosynthetic reaction steps. Preliminary data obtained from needles of a healthy and a declining spruce as monitored during the growing season 1994 are presented. Light emission of delayed light as observed both within 500 ms and within 30 s time periods is interpreted in terms of "vitality."     

Vibrational energy relaxation of naphthalene in the S(1) state in various gases

Time-resolved fluorescence spectra of naphthalene in the S(1) state have been measured in various gases below 10(2) kPa. The band shape of the fluorescence changed in an earlier time region after the photoexcitation when an excess energy (3300 cm(-1)) above the 0-0 transition energy was given. The excitation energy dependence of the fluorescence band shape of an isolated naphthalene molecule was measured separately, and the time dependence of the fluorescence band shape in gases was found to be due to the vibrational energy relaxation in the S(1) state. We have succeeded in determining the transient excess vibrational energy by comparing the time-resolved fluorescence band shape with the excitation energy dependence of the fluorescence band shape. The excess vibrational energy decayed almost exponentially. From the slope of the decay rate against the buffer gas pressure, we have determined the collisional decay rate of the excess vibrational energy in various gases. The dependence of the vibrational energy relaxation rate on the buffer gas species was similar to the case of azulene. The comparisons with the results in the low temperature argon and the energy relaxation rate in the S(0) state in nitrogen were also discussed.     

DELAYED MICROSECOND LUMINESCENCE OF PHYCOBILISOMES

The time-resolved luminescence spectra (in the microsecond range) of phycobilisomes and biliproteins in buffer and polymer matrix were measured in the temperature range from 8 K. to 293 K. Delayed luminescence located in the same spectral region as prompt fluorescence of investigated samples (DLF) and the long-wavelength delayed emission in the720–760 nm range (DL1) was observed. The temperature and viscosity dependencies of DLF and DL1 luminescences were different, but both do not have uniexponential decays and are not quenched by oxygen. This means that delayed luminescence could be generated without the participation of the triplet states, or the chromophores could be shielded by protein against interaction with oxygen. The linear dependence of delayed luminescence on exciting light intensity shows that delayed luminescence is monophotonically induced. It seems that both DLF and DL1 are related to electron-cation recombination, which yields excited singlet states. The DLF is emitted from the first excited singlet state of biliprotein chromophores and DL1 from the same state of the excimers or from the triplet state of some groups of chromophores. Ionization energy of chromophores can be lowered as a result of their interactions with the environment. Delay of emission is due to the trapping or solvation of electrons. Every type of biliprotein consisting of phycobilisomes possesses its own “trap” and can emit the DL. In the case of native phycobilisomes a competition between the excitation energy trapping and transfer occurs.