Luminescence quenching by nitroxide spin labels in aqueous solution: Studies on the mechanism of quenching

Janos Matko, Kazuo Ohki, Michael A Edidin

Research output: Contribution to journalArticle

Abstract

The mechanism of luminescence quenching by spin labels was investigated in aqueous solution by steady-state and time-resolved luminescence techniques. Water-soluble nitroxide radicals strongly quenched the luminescence emitted by Tb3+ chelates and by fluorescein, either free or conjugated to proteins. The following features of the quenching reaction were established: (I) the rate constant for quenching of triplet-state Tb3+ by nitroxides was about 4 orders of magnitude smaller (ca. 105 M-1 s-1) than those of the singlet-state probes; (H) the quenchers reduced the excited-state lifetime of both probes; (III) the rate constants for quenching of both probes were found to be apparently independent of the temperature (between 6 and 42°C) and viscosity (up to 60 mPa·s) of the solutions; (IV) both singlet and triplet quenching rates were sensitive to solvent polarity; (V) there is a small but significant spectral overlap between the absorption band of weekly absorbing nitroxide radicals and the emission spectra of luminophores, the extent of which, however, does not correlate with the extent of quenching; (VI) the quenching rate declines sharply with an increasing luminophore to nitroxide distance. The distance dependence of the quenching rate showed a satisfactory fit to an exponential function. These findings indicate that the quenching reaction is dominated by an electron exchange between the excited singlet- or triplet-state luminophore and the nitroxide radical rather than controlled by diffusional properties of the reactants. The extremely high sensitivity of the quenching rate to the separation distance in the range of 0.5-2 nm should make nitroxide-conjugated antibodies paired with fluorophore-conjugated antibodies a useful fluorophore-quencher system for studying lateral organization and association of membrane proteins on cell surfaces.

Original languageEnglish (US)
Pages (from-to)703-711
Number of pages9
JournalBiochemistry®
Volume31
Issue number3
StatePublished - 1992

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Spin Labels
Luminescence
Quenching
Membrane Proteins
Antibodies
Fluorescein
Viscosity
Electrons
Fluorophores
Temperature
Water
Rate constants
nitroxyl
Proteins
Exponential functions
Excited states
Absorption spectra
Association reactions

ASJC Scopus subject areas

  • Biochemistry

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Luminescence quenching by nitroxide spin labels in aqueous solution : Studies on the mechanism of quenching. / Matko, Janos; Ohki, Kazuo; Edidin, Michael A.

In: Biochemistry®, Vol. 31, No. 3, 1992, p. 703-711.

Research output: Contribution to journalArticle

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