Further Observations on the Measurement of Fatty Acid Incorporation by Erythrocyte Ghosts to Quantify Unbound Palmitate Concentration in Albumin–Palmitate Mixtures

Abstract

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°C). A stoichiometric binding constant for 1:1 complex formation (K1) of 4.6 (±0.3) × 108M−1was obtained from experiments in which erythrocyte ghosts were the source of fatty acid. An essentially identical estimate of 4.1 (±0.7) × 108M−1was 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 K1estimate, 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.