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

✍ Scribed by Susan M. Pond; Rosalind A. Gordon; Anne L. Simi; Donald J. Winzor

• Publisher: Elsevier Science
• Year: 1996
• Tongue: English
• Weight: 121 KB
• Volume: 237
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1996.0234

✦ Synopsis

acids. Although fatty acids interact with many sites

Advantage has been taken of the extensive and reon albumin, they bind very strongly to a hydrophobic versible incorporation of long-chain fatty acids by channel in subdomain IIIA (1), at each end of which is erythrocyte ghosts to characterize the interaction of a cluster of basic residues that could be interacting tritium-labeled palmitic acid with human serum alwith the carboxyl group of the fatty acid (2-5).

bumin (pH 7.4, 37ЊC). A stoichiometric binding con-

Because the proportion of unbound long-chain fatty stant for 1:1 complex formation (K 1 ) of 4.6 ({0.3) 1 10 8 acid molecules is only in the vicinity of 0.01% (6), M 01 was obtained from experiments in which erythroit is not surprising that precise measurement of the cyte ghosts were the source of fatty acid. An essenunbound concentration of these ligands has proven tially identical estimate of 4.1 ({0.7) 1 10 8 M 01 was very difficult (7, 8). Most quantitative studies of the obtained from a second series of experiments in fatty acid/albumin interaction have entailed examiwhich the [ 3 H]palmitate was included with the albunation of the effect of protein on the partition of fatty min in the aqueous phase. The magnitude of the presacid between aqueous and n-heptane phases (6, 9ent K 1 estimate, which is three-to fivefold larger than 13). However, advantage has also been taken of the most recently reported values, reflects binding mearapid and extensive incorporation of long-chain fatty surements restricted to a very limited range of unacids into erythrocyte ghosts (14 -17) to measure the bound palmitate concentration (00.2 nM) to ensure concentration of unbound palmitate in the presence that the ligand is essentially monomeric. This use of of albumin. Although similar to the partition method erythrocyte ghosts to quantify the palmitate -albuinasmuch as the effect of albumin on the distribution min interaction has reinforced the basic tenets of a of fatty acid between phases is being measured, the published procedure [I. N. Bojesen, and E. Bojesen erythrocyte ghost procedure differs because the incor-(1992) J. Lipid Res. 33, 1327 -1334], the major virtue of which is its ability to provide a direct measure of poration of fatty acid by the ghosts reflects binding to the equilibrium concentration of unbound fatty acid specific sites rather than partition into the erythroin albumin -palmitate mixtures.