Further observations on the measurement of fatty acid incorporation by erythrocyte ghosts to quantify unbound palmitate concentration in albumin-palmitate mixtures.

Advantage has been taken of the extensive and reversible incorporation of long-chain fatty acids by erythrocyte ghosts to characterize the interaction of tritium-labeled palmitic acid with human serum albumin (pH 7.4, 37 degrees C). A stoichiometric binding constant for 1:1 complex formation (K1) of 4.6 (+/-0.3) x 10(8) M-1 was obtained from experiments in which erythrocyte ghosts were the source of fatty acid. An essentially identical estimate of 4.1 (+/-0.7) x 10(8) M-1 was obtained from a second series of experiments in which the [3H]palmitate was included with the albumin in the aqueous phase. The magnitude of the present K1 estimate, which is three- to fivefold larger than most recently reported values, reflects binding measurements restricted to a very limited range of unbound palmitate concentration (-0.2 nM) to ensure that the ligand is essentially monomeric. This use of erythrocyte ghosts to quantify the palmitate-albumin interaction has reinforced the basic tenets of a published procedure [I. N. Bojesen, and E. Bojesen (1992) J. Lipid Res. 33, 1327-1334], the major virtue of which is its ability to provide a direct measure of the equilibrium concentration of unbound fatty acid in albumin-palmitate mixtures.