Interactions of long-chain fatty acids and albumin: Determination of free fatty acid levels using the fluorescent probe ADIFAB

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 OC. Under conditions observed in normal human physiology (v I 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 (-3000 nM), for example, were observed for linolenate (1 8:3) 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. For example, overall, affinities for murine albumin were lower (higher (FFA) values) than for human and bovine albumins. Specific differences were also observed in the relative affinities of the three albumins for different FA. Oleate binds more tightly than palmitate to human and murine albumins, while the reverse is true for bovine. The measured binding isotherms are in all cases well described by a multiple stepwise equilibrium model with six association constants. In most cases, the association constant for the first site is greater than or equal to that of succeeding sites. In a few instances, however, in particular for arachidonate binding to human albumin, the results (a smaller constant for the first than the second site) suggest that binding may involve positive cooperativity. It is also shown that the binding isotherms generated by the multiple stepwise model, are themselves well described by a linear plus exponential function, thereby allowing FFA levels to be estimated by simple calculation.