Location of the medium chain fatty acid site on human serum albumin. Residues involved and relationship to the indole site.

Abstract

Human serum albumin (HSA) is found to contain one primary binding site for medium chain fatty acids which is competitive with the binding of N-acetyl-L-tryptophan. The association constant is higher with nondefatted HSA than with HSA defatted by either heptane-acetic acid or charcoal extraction. Affinity labeling of HSA by N-bromoacetyl-n-decylamine (1:1 molar ratio) blocks the primary binding site of hexanoate and decanoate at an equivalency of the molar ratio of label incorporated. CNBr fragmentation reveals that the primary medium chain fatty acid binding site is positioned between Fragment C (residues 124 to 297) and A (residues 298 to 585). With nondefatted HSA, 60% of the label is associated with Fragment C and 35% with Fragment A, whereas upon defatting, this labeling pattern is reversed. The labeled residue in Fragment C is Lys 199, the same residue as acetylated by acetylsalicylate (Hawkins, D., Pinckard, R.N., Crawford, I.P., and Farr, R.S. (1969) J. Clin. Invest. 48, 536). Forty per cent of the label in Fragment A from nondefatted albumin is located in the A-Pro II subfragment (residues 447 to 548) and 60% in the A-Phe subfragment (residues 330 to 446). With defatted HSA, the A-Pro II subfragment contains 25% and the A-Phe subfragment contains 75% of the label. In all cases, labeling in the subfragments of Fragment A occurred at several different residues, indicating that this part of the binding site has considerable flexibility. The secondary binding sites for medium chain fatty acid are found to be the same general region as the primary binding site. N-Bromoacetyl-n-tetradecylamine (a long chain fatty acid-labeling agent) labeled only the A fragment of HSA.