Quenching of fluorescence by triplet excited states in chloroplasts

Jacques Breton, Nicholas Geacintov, Charles E. Swenberg

Research output: Contribution to journalArticle

Abstract

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5-4.0 μs duration dye laser flash of varying intensities as an excitation source. The yield (F{cyrillic}) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectroscopy at 515 nm, while the yield F{cyrillic} following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10-12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from F{cyrillic} utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (〉1016 photon · cm-2) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1-2 μs after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules. At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse. A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a singlet-triplet bimolecular rate constant of γst = 10-8 cm3 · s-1, the following observations can be accounted for: (1) the rapid initial drop in F{cyrillic} and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.

Original languageEnglish (US)
Pages (from-to)616-635
Number of pages20
JournalBBA - Bioenergetics
Volume548
Issue number3
DOIs
StatePublished - Dec 6 1979

Fingerprint

Chloroplasts
Carotenoids
Excited states
Quenching
Lasers
Fluorescence
Laser pulses
Chlorophyll
Molecules
Kinetics
Ions
Xenon
Dye lasers
Dye Lasers
Quantum yield
Spinacia oleracea
Absorption spectroscopy
Photons
Rate constants
Differential equations

Keywords

  • Carotenoid
  • Chlorophyll
  • Chloroplast
  • Fluorescence quenching
  • Triplet state

ASJC Scopus subject areas

  • Biophysics
  • Medicine(all)

Cite this

Quenching of fluorescence by triplet excited states in chloroplasts. / Breton, Jacques; Geacintov, Nicholas; Swenberg, Charles E.

In: BBA - Bioenergetics, Vol. 548, No. 3, 06.12.1979, p. 616-635.

Research output: Contribution to journalArticle

Breton, Jacques ; Geacintov, Nicholas ; Swenberg, Charles E. / Quenching of fluorescence by triplet excited states in chloroplasts. In: BBA - Bioenergetics. 1979 ; Vol. 548, No. 3. pp. 616-635.
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