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European Biophysics Journal

, Volume 28, Issue 6, pp 526–531 | Cite as

Thermal stability of a flavoprotein assessed from associative analysis of polarized time-resolved fluorescence spectroscopy

  • A. V. Digris
  • V. V. Skakoun
  • E. G. Novikov
  • A. van Hoek
  • A. Claiborne
  • A.J. W. G. Visser
BIOPHYSICS LETTER

Abstract

Upon gradually heating a particular mutant of the flavoprotein NADH peroxidase, it was found from the peculiar time-resolved fluorescence anisotropy pattern of the flavin prosthetic group (FAD) that, at elevated temperature, FAD is released from the tetrameric enzyme. Since in this case a mixture of free and enzyme-bound FAD contributes to the time-dependent fluorescence anisotropy, its analysis can only be accomplished by an associative fitting model, in which specific fluorescence lifetimes of both species are linked to specific correlation times. In this letter the general approach to the associative polarized fluorescence decay analysis is described. The procedure can be used for other flavoproteins to determine the temperature at which the onset of thermal denaturation will start, leading to release of the flavin prosthetic group.

Key words Time-resolved fluorescence Fluorescence anisotropy decay Global analysis Associative fitting model 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1999

Authors and Affiliations

  • A. V. Digris
    • 1
  • V. V. Skakoun
    • 1
  • E. G. Novikov
    • 1
  • A. van Hoek
    • 2
  • A. Claiborne
    • 3
  • A.J. W. G. Visser
    • 2
  1. 1.Systems Analysis Department, Belarusian State University, Minsk 220050, Belarus
  2. 2.MicroSpectroscopy Center, Department of Biomolecular Sciences, Wageningen Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands e-mail: ton.visser@laser.bc.wau.nlNL
  3. 3.Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, NC 27157, USAUS

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