Purification and Fluorescent Titration of Cellular Retinol-Binding Protein

The intracellular carriers for all-trans retinol are believed to be the homologous cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) (ref. 1 , and references therein). CRBP is present in a wide variety of tissues, including liver, kidney, and testis. The basic feature of CRBP is the retinol-protein recognition, through which a drastically higher stability for CRBP-bound retinol relative to uncomplexed retinol is achieved. In addition, the binding to CRBP permits the solubilization in the aqueous medium of the highly hydrophobic retinol molecule. Evidence has been presented to indicate that the retinol-CRBP complex may serve as substrate of enzymes involved in the metabolism of retinol (2 –4 ). The binding of retinol to CRBP is characterized by a dissociation constant lower than nanomolar (5 , 6 ). CRBP exhibits affinity for a variety of retinol analogs (6 , 7 ). However, the binding of retinol analogs to CRBP was found to be substantially weaker than that of retinol (6 ). Based on the three-dimensional structure determination of the retinol-CRBP complex (8 ) and on the results of studies performed in solution (6 ), it has been suggested that the hydrogen-bonding interaction between the retinol-hydroxyl group and the side chain of Gln 108 contributes remarkably to the strength of binding of retinol to CRBP.