Equilibrium binding of long-chain fatty acids (FA) with albumin from human serum (HSA), bovine serum (BSA), and murine serum (MSA) has been studied by measuring the equilibrium levels of free fatty acids (FFA). FFA levels were measured directly, using a new fluorescent probe composed of acrylodan-derivatized intestinal fatty acid binding protein (ADIFAB). Measurements of [FFA] were done as a function of the ratio of total FA to total albumin (v) for v values between 0 and 6, at pH 7.4 and 37 degrees C. Under conditions observed in normal human physiology (v < or = 2), [FFA] values of the most abundant serum FA (palmitate, stearate, oleate) in equilibrium with human or bovine albumin are less than 15 nM. These values are considerably smaller than the generally quoted values of [FFA] in equilibrium with albumin: more than 20-fold for palmitate and more than 50-fold for oleate. FFA levels were found to increase monotonically with for all three albumins and all FA. In most cases [FFA] increased, for the same chain length, with increasing degree of acyl chain unsaturation, suggesting that FA aqueous solubility may play a significant role in the equilibrium between FA association with albumin and the aqueous phase. [The highest FFA levels (approximately 3000 nM), for example, were observed for linoleate (18:3) at the maximum v value (6).] Although aqueous-phase solubility of the FA may be important in understanding the interaction between FA and albumin, protein structure, as reflected in differences among the three albumins, also significantly affects the equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)