INTERACTIONS OF LONG-CHAIN FATTY-ACIDS AND ALBUMIN - DETERMINATION OFFREE FATTY-ACID LEVELS USING THE FLUORESCENT-PROBE ADIFAB

Equilibrium binding of long-chain fatty acids (FA) with albumin from h uman 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 comp osed of acrylodan-derivatized intestinal fatty acid binding protein (A DIFAB). 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 less-than-or-equal-to 2), [FFA] values of the most abundant serum FA (palmitate, stearate, oleate) in equilibrium with human or bovine a lbumin are less than 15 nM. These values are considerably smaller than the generally quoted values of [FFA] in equilibrium with albumin: mor e than 20-fold for palmitate and more than 50-fold for oleate. FFA lev els were found to increase monotonically with for all three albumins a nd all FA. In most cases [FFA] increased, for the same chain length, w ith increasing degree of acyl chain unsaturation, suggesting that FA a queous solubility may play a significant role in the equilibrium betwe en FA association with albumin and the aqueous phase. [The highest FFA levels (approximately 3000 nM), for example, were observed for linole nate (18:3) at the maximum v value (6).] Although aqueous-phase solubi lity of the FA may be important in understanding the interaction betwe en FA and albumin, protein structure, as reflected in differences amon g the three albumins, also significantly affects the equilibrium. For example, overall, affinities for murine albumin were lower (higher [FF A] values) than for human and bovine albumins. Specific differences we re also observed in the relative affinities of the three albumins for different FA. Oleate binds more tightly than palmitate to human and mu rine albumins, while the reverse is true for bovine. The measured bind ing isotherms are in all cases well described by a multiple stepwise e quilibrium model with six association constants. In most cases, the as sociation constant for the first site is greater than or equal to that of succeeding sites. In a few instances, however, in particular for a rachidonate binding to human albumin, the results (a smaller constant for the first than the second site) suggest that binding may involve p ositive cooperativity. It is also shown that the binding isotherms gen erated by the multiple stepwise model, are themselves well described b y a linear plus exponential function, thereby allowing FFA levels to b e estimated by simple calculation.