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 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 (28)Leu 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.
Donato
Leslie J
LJ
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
Noy
Noa
N
D000042:Absorption; D000818:Animals; D016477:Artifacts; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D002138:Calibration; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D011487:Protein Conformation / Q000187:drug effects; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014212:Tretinoin / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology
D005456:Fluorescent Dyes; D008024:Ligands; D018168:Receptors, Retinoic Acid; C082927:retinoic acid binding protein I, cellular; D014212:Tretinoin