Fluorescence anisotropy of fluorescein derivative varies according to pH: Lessons for binding studies

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Abstract

Fluorescence spectroscopy is a powerful tool to comprehend macromolecules interaction. The characteristics of the fluorophore as well its behavior under the experimental conditions are central to ascertain the results. The present work characterizes the fluorescence emission and anisotropy of a fluorescein derivative linked to a double strand DNA containing 67 base pairs (named as 6-FAM-67-mer) under different pH conditions. The results show that fluorescence emission and anisotropy of this fluorescein derivative varies according to pH (as the pH becomes more acidic, the total fluorescence emission decreases). The decrease in fluorescence emission intensity is accompanied by an increase in anisotropy. The monoanionic form of fluorescein derivative is the predominant species from pH 7.5 to 9.2. Our main conclusion is that a constant fluorescein derivative emission is a prerequisite to determine association constants between macromolecules.

Original languageEnglish
Pages (from-to)59-62
Number of pages4
JournalJournal of Photochemistry and Photobiology A: Chemistry
Volume372
DOIs
Publication statusPublished - 1 Mar 2019

Keywords

  • 6-FAM
  • Anisotropy
  • dsDNA
  • Fluorescein derivative
  • Fluorescence spectroscopy

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