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Retinoids pp 177-187 | Cite as

Fluorescence-Based Technique for Analyzing Retinoic Acid

  • Leslie J. Donato
  • Noa Noy
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 652)

Abstract

Retinoic acid (RA) is a potent transcriptional activator whose actions are mediated by members of the nuclear hormone receptor family. In addition to playing key roles in embryonic development and in tissue maintenance in the adult, RA is a potent anticarcinogenic agent currently in clinical use for treatment of various cancers. Here, we describe an optical method for measuring the concentrations of RA in biological samples. This method uses cellular retinoic acid-binding protein I (CRABP-I), a protein that binds RA with high affinity and specificity, as a “read-out” for its ligand. Replacing 28Leu of CRABP-I with a Cys residue allows for covalently attaching an environmentally sensitive fluorescent probe to the protein at a region that undergoes a significant conformational change upon ligand binding. Association of RA with the modified protein thus results in changes in the fluorescence of the probe, enabling reliable measurements of RA concentrations as low as 50 nM. We show that the method can be effectively used to measure RA concentrations in serum and to monitor the biosynthesis and the degradation of RA in cultured mammalian cells.

Key words

Retinoic acid intracellular lipid-binding proteins retinoic acid-binding protein equilibrium dissociation constant 5-bromomethyl fluorescein fluorescence titration retinoic acid biosynthesis retinoic acid degradation 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Leslie J. Donato
    • 1
  • Noa Noy
    • 2
  1. 1.Department of BiochemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Departments of Pharmacology and NutritionCase Western Reserve University School of MedicineClevelandUSA

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