Changes in the room-temperature emission spectrum of chlorophyll during fast and slow phases of the Kautsky effect in intact leaves

in Photochemistry & Photobiology (2005), 81(2, Mar-Apr), 431-436

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves ... [more ▼]

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F-O (basal level of fluorescence yield at transient O) and F-M (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F-O to F-M, a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F-M to FT (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength-emitting Chl ascribed to LHCII. [less ▲]

Spectroscopic properties of protochlorophyllide analyzed in situ in the course of etiolation and in illuminated leaves

in Photochemistry & Photobiology (2000), 72(1), 85-93

The spectroscopic properties of photoactive (ie. flash-transformable) and nonphotoactive protochlorophyll(ide)s (Pchl(ide)) were reinvestigated during the development of bean leaves in darkness. Two ... [more ▼]

The spectroscopic properties of photoactive (ie. flash-transformable) and nonphotoactive protochlorophyll(ide)s (Pchl(ide)) were reinvestigated during the development of bean leaves in darkness. Two phases in the process of Pchl(ide) accumulation were apparent from quantitative measurements of pigment content: a lag phase (first week) during which photoactive Pchl(ide) accumulated faster than nonphotoactive Pchl(ide); and a fast phase (second week), showing parallel accumulation of both types of Pchl(ide). 'Flashed-minus-dark' absorbance difference spectra recorded in situ at 77 K showed that P650-655 was the predominant form of photoactive protochlorophyllide regardless of developmental stage. Quantitative analysis of energy migration processes between the Pchl(ide) forms showed the existence of energy transfer units containing a 1:8 ratio of nonphotoactive and photoactive Pchl(ide)s during development. Gaussian deconvolution of in situ 77 K fluorescence spectra indicated that the 633 nm band of nonphotoactive Pchl(ide) was made of four bands, at 625, 631, 637 and 643 nm, whose relative amplitudes only slightly changed during development. The emission band of photoactive Pchlide was also analyzed using the same method. Three components were found at 644, 652 and 657 nm, The emission band of P650-655 included the last two components, which become predominant only in fully etiolated plants. Photoactive Pchlide with an emission maximum at 653 nm was detected in the light during development of leaves of photoperiodically grown plants. [less ▲]

Analysis of fluorescence lifetime of protochlorophyllide and chlorophyllide in isolated etioplast membranes measured from multifrequency cross-correlation phase fluorometry

in Photochemistry & Photobiology (1999), 70(4), 616-623

The fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different ... [more ▼]

The fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple-exponential model at 14 degrees C and a double-exponential model at -170 degrees C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14 degrees C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different Pchlide forms in steady-state fluorescence spectra, The fluorescence decay of the main Pchlide form, photoactive Pchlide-F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5-6.0 ns are ascribed to the second photoactive form, Pchlide-F645, and to nonphotoactive Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating fight pulse, we found a single 5.0 ns component for Chlide-F688 (the Chlide-NADPH :protochlorophyllide oxidoreductase [POR]-NADP(+) complex) and an additional 1.6 ns component when the formation of Chlide-F696 (the Chlide-POR-NADPH complex) was promoted by exogenous NADPH, From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide-F696 as being 70%. [less ▲]

Electron Spin Resonance Evidence of the Generation of Superoxide Anion, Hydroxyl Radical and Singlet Oxygen During the Photohemolysis of Human Erythrocytes with Bacteriochlorin A

in Photochemistry & Photobiology (1997), 66(4), 502-8

Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a ... [more ▼]

Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate-buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm2. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K-leakage and hemolysis suggesting a type II mechanism. The D2O did not affect K-leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K-leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin-trapping techniques that BCA is able to generate O2-. and OH. radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct DMPO-OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH3CHOH. and CO2-. radical adducts, indicating the presence of free OH.. Production of DMPO-OH was partly inhibited by superoxide dismutase (SOD), catalase and desferrioxamine, suggesting that the iron-catalyzed decomposition of H2O2 was partly involved in the formation of one part of the observed OH.. The complementary inhibition of DMPO-OH production by azide and 9,10-anthracenedipropionic acid (ADPA) was consistent with 1O2 production by BCA followed by reaction of 1O2 with DMPO and decay of the intermediate complex to form DMPO-OH and free OH.. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye-induced hemolysis of erythrocytes was cell caused by a mixed type 1/type 2 mechanism. [less ▲]

On the charge transfer pathway in the Merocyanine 540 triplet state quenching by nitroxyl radical

in Photochemistry & Photobiology (1995), 61

Photoionization of tryptophan: An electron spin resonnance investigation

in Photochemistry & Photobiology (1986), 44

A short-lived intermediate in the photoenzymatic reduction of protochlorophyll(ide) into chlorophyll(ide) at a physiological temperature

in Photochemistry & Photobiology (1980), 32

The reduction of protochlorophyll(ide) into chlorophyll(ide) has been studied by flash absorption spectroscopy at 21°C, with a time resolution of 0.5 µs. The absorption changes have been recorded in the ... [more ▼]

The reduction of protochlorophyll(ide) into chlorophyll(ide) has been studied by flash absorption spectroscopy at 21°C, with a time resolution of 0.5 µs. The absorption changes have been recorded in the range 670–720 nm after the first and subsequent flashes given to an extract of etiolated bean leaves. At 695 nm the flash-induced absorption increase has its maximum value immediately after the flash and then partly decays with a half-time of about 7–10 µs. A complementary behaviour is observed at 675 nm where the absorption change is very small at time zero and then increases to a stationary value with a half-time of about 6–9 µs. From measurements at several wavelengths it is concluded that a species with an absorption peak around 695 nm is formed immediately after the flash and is then transformed into a stable species with an absorption peak around 675 nm. Measurements at lower temperatures, down to—50°C, show that the transformation is much slowed down at decreased temperatures. The species absorbing at 695 nm (P695) is attributed to an intermediate in the photoreduction of protochlorophyll(ide) P639,650 into chlorophyll(ide) P676. When the protochlorophyll(ide) is photoreduced before the flash illumination, the newly formed chlorophyll(ide) gets to a triplet state, which decays with a half-time of 15 µs at 21°C. This result shows that carotenoid molecules do not exert their protective role at this stage of chlorophyll (Chl) formation [less ▲]