The binding properties of rat h e r fatty acid - binding protein

Fatty acid-binding protein (FABP) from rat liver cytosol is an abundant 14 000-M, monomeric protein that is known to have a high affinity for both fatty acids and their CoA esters, as well as a number of non-polar organic anions (Glatz & Veerkamp, 1985). The precise physiological role of this protein remains unclear and clarification of its functions will require more precise information on its ligand-binding properties, the number of binding sites and the affinity of the protein for the various types of ligand. We have recently reported that the fluorescent fatty acid probe, dansylundecanoic acid, binds to rat liver FABP with high affinity and shows a considerable fluorescence enhancement (@fold) compared with the probe in buffer (Wilkinson & Wilton, 1986). As a result of these properties, we have used this probe to detect and quantify rat liver FABP in biological samples (Wilkinson & Wilton, 1986). In the present work, we have used this fluorescent probe to examine the binding properties of FABP in vitro in more detail. Dansylundecanoic acid shows a considerable fluorescence enhancement and spectral shift on binding to FABP and the emission maximum (500 nm) of the protein-bound probe is the same as that observed when the probe binds to albumin. This is consistent with the probe binding in a non-polar environment. The probe can be completely displaced from FABP by the addition of an excess of long-chain fatty acid (e.g. oleic acid) whilst dansyl glycine, which lacks the acyl chain of dansyl undecanoic acid, shows no significant binding over the concentration range tested. Fluorescence titration experiments performed using the probe with purified FABP shows saturable binding of dansylundecanoic acid to a single binding site per molecule of protein as assessed by Scatchard analysis. This stoichiometry is consistent with the value obtained for the binding of long-chain fatty acids to rat liver FABP assessed using the Lipidex 1000 method (Glatz et al., 1984; Bass, 1985). The affinity of dansylundecanoic acid binding to FABP was assessed by plotting binding data according to the method of Scatchard (Scatchard, 1949). Surprisingly, Scathcard analysis of the binding gave a curved plot (Fig. 1) which could be resolved into a highand low-affinity binding form of the protein (dissociation constants 0.03 PM and 0.5 PM, respectively), the relative proportions of the two forms varying with the concentration of FABP in the assay. We believe this complex behaviour with respect to binding of the fluorescent probe may be due to the existence of two or more interchangeable conformations of the protein which are detectable because of the rapid nature of the fluorescence assay. These conformational states may be equivalent to the various interconvertible isoforms of the protein reported by