Fluorometric Titration of the CRABPs
Fluorescence spectroscopy has long been used to characterize the equilibrium binding of retinoids to proteins (1). Changes in steady-state fluorescence are monitored as the protein is titrated with aliquots of retinoid. The resultant binding curve can be analyzed yielding information about the stoichiometry and affinity of retinoid binding. There are several advantages to this approach. For one, this is a true equilibrium technique. Binding is monitored directly, with no need to physically separate bound from free ligand. A second advantage is the high sensitivity of fluorescence. Titrations can be routinely performed on as little as 2.5 mL of 10−6-10−7 M CRABP. Another advantage is that no alteration of the retinoid or protein is necessary. The fluorescence signals are intrinsic to the native protein and retinoid.
KeywordsRetinoic Acid Fluorescence Signal Sigma Plot Filter Effect Unbind Concentration