Fluorescence-Based Technique for Analyzing Retinoic Acid

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