Thermodynamic and Kinetic Properties of Fatty Acid Interactions with Rat Liver Fatty Acid-binding Protein

Fatty acid-binding protein from rat liver (L-FABP) binds 2 fatty acids (FA) per protein, in contrast to FABPs from adipocyte, heart, and intestine, for which binding and structural studies are consistent with a single FA binding site. To understand better the unique characteristics of L-FABP, we have carried out equilibrium binding and kinetic measurements of long chain FA using the fluorescent probes of free fatty acids (FFA), ADIFAB and ADIFAB2, to monitor the concentration of FFA in the reaction of FA with L-FABP. We found that the dissociation constants (Kd) ranged from about 1 nM to 4 μM, being largest for myristate at 45°C and smallest for oleate at 10°C, and that 2 FA were bound per L-FABP for all temperatures and FA. The binding measurements also revealed that at temperatures below 37°C, affinities for the two binding sites differ by between 5- and 20-fold but as the temperature was increased, the affinities converge toward equal values. Off-rate constants (koff) were similar for all FA and for temperatures between 10 and 45°C, ranged from about 0.1 s−1 to 50 s−1. Moreover, for all FA, koff values for dissociation from both the high and low affinity sites were similar, indicating that binding affinity differences at the lower temperatures reflect lower on-rates for binding to the low affinity site. The temperature at which the affinities of the two sites become equivalent depends upon the FA; higher temperatures (45-50°C) are required for the unsaturated FA and myristate than for the longer chain saturated FA (