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

• Published in: Journal of photochemistry and photobiology. A, Chemistry. Chemistry., 2019-03, Vol.372, p.59-62
• Main Authors: Castro, Nathália S.S., Moura, Isabel, Carepo, Marta S.P., Laia, César A.T.
• Format: Article
• Language: English
• Subjects: 6-FAM; Anisotropy; Base pairs; Deoxyribonucleic acid; DNA; dsDNA; Emissions; Fluorescein; Fluorescein derivative; Fluorescence; Fluorescence spectroscopy; Macromolecules; pH effects
• ISSN: 1010-6030; 1873-2666
• DOI: 10.1016/j.jphotochem.2018.12.008

[Display omitted] •Fluorescence emission of a fluorescein derivative decreased in acid pH.•Fluorescence anisotropy of fluorescein derivative increased in acid pH.•The monoanionic species of fluorescein derivative predominates under physiological pH.•A constant fluorescence emission of fluorescein derivative is a prerequisite for binding studies. 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.