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Journal of Lipid Research, Vol. 10, 56-67, January 1969
Copyright © 1969 by Lipid Research, Inc.

Binding of long-chain fatty acids to bovine serum albumin

Arthur A. Spector , Kathryn John , and John E. Fletcher

Laboratory of Metabolism, National Heart Institute, and Laboratory of Applied Studies, Division of Computer Research Technology, National Institutes of Health, Bethesda, Maryland 20014

We have studied the binding of long-chain free fatty acids (FFA) to crystalline bovine serum albumin (BSA) that had been extracted with charcoal to remove endogenous fatty acids. The data were analyzed in terms of a model consisting of six high-energy binding sites and a large number of weak binding sites.

The high-energy sites were resolved into two distinct classes, each containing three sites. At 37°C and pH 7.4, k'₁ (the apparent association constant of a class of binding sites) was about 10⁶ m^-1 for binding to the three primary sites, and k'₂ was about 10⁵ m^-1 for binding to the three secondary sites. The number of weak (tertiary) sites was estimated to be 63 with a k'₃ of 10³ m^-1. In general, palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, or myristate, and much more tightly than laurate.

The association of palmitate with human and rabbit albumin also was analyzed in terms of this model. Palmitate was bound less firmly by human or rabbit albumin than by BSA. Palmitate binding to BSA was dependent upon the pH and temperature of the incubation medium.

Long-chain hydrocarbons that did not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexadecane) were bound to a limited extent and weakly. The presence of positively charged protein sites and native protein tertiary structure were required for maximal binding of palmitate to BSA. Of nine other proteins tested, only ßbeta;-lactoglobulin exhibited a significant capacity to bind palmitate.

Supplementary key words free fatty acids • association constants • pH • temperature • hexadecane • methyl palmitate • cetyl alcohol • ßbeta;-lactoglobulin

Submitted on January 30, 1968
Accepted on September 23, 1968

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