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Nanosecond Time-Resolved Fluorescence Studies of Thioredoxin

  • M. Han
  • M. VandeVen
  • D. Walbridge
  • J. Knutson
  • R. Lessick
  • C. Anfinsen
  • L. Brand
Conference paper

Abstract

Thioredoxin from E. coli contains two tryptophan residues, trp 28 and trp 31, close to two cysteine residues, cys 32 and cys 35, which form a disulfide bond. Time-resolved fluorescence decay of tryptophan was monitored at emission wavelength of 340 nm with excitation wavelength of 295 nm in 100 mM potassium phosphate buffer, pH 5.7, and 1 mM EDTA at 5°C. Oxidized thioredoxin exhibits a triple fluorescence decay with lifetimes of 0.3, 1.4, and 5.9 nsec. Upon reduction of the disulfide bond by dithiothreitol (DTT), the steady-state fluorescence intensity increased and decay times of 0.4, 2.4, and 5 ns were found. The kinetics of reduction were also studied with a pulse fluorometer by collecting thirty consecutive decay curves at a rate of 2 min per curve for one hour under computer control. Simultaneous global analysis of the data, with lifetimes linked, indicates that the amplitude associated with the longest lifetime increases significantly, while the amplitude associated with the short lifetime decreases. Rate constants for the amplitude changes with time were estimated by a non-linear least squares method. Enzyme denatured by guanidine-HCl shows increased fluorescence. The fluorescence decay exhibits a triple exponential decay with lifetimes of 0.4, 2.0, and 4.4 nsec. Addition of DTT to the denatured enzyme does not increase the fluorescence intensity nor change the lifetimes. The time-resolved emission anisotropy of both reduced and oxidized enzyme was also studied.

Keywords

Disulfide Bond Decay Time Emission Wavelength Cysteine Residue Potassium Phosphate Buffer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • M. Han
    • 1
    • 2
  • M. VandeVen
    • 1
    • 2
  • D. Walbridge
    • 1
    • 2
  • J. Knutson
    • 1
    • 2
  • R. Lessick
    • 1
    • 2
  • C. Anfinsen
    • 1
    • 2
  • L. Brand
    • 1
    • 2
  1. 1.Department of VirologyThe Johns Hopkins UniversityBaltimoreUSA
  2. 2.The Laboratory of Technical DevelopmentNHLBI, NIHBethesdaUSA

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