Abstract
The remarkable binding properties of serum albumin have been investigated extensively, but little is known about an important class of fatty acids, the very long-chain saturated fatty acids (VLCFA; >18 carbons). Although VLCFA are metabolized efficiently in normal individuals, they are markers for and possibly causative agents of several peroxisomal disorders. We studied the binding of [13C]carboxyl-enriched arachidic (C20:0), behenic (C22:0), lignoceric (C24:0), and hexacosanoic (C26:0) acids to bovine serum albumin (BSA) by 13C-NMR spectroscopy. For each VLCFA, the NMR spectra showed multiple signals at chemical shifts previously identified for long-chain fatty acids (12–18 carbons), suggesting stabilization of binding by similar, if not identical, interactions of the fatty acid carboxyl anion with basic amino acid residues. The maximal binding (mol of VLCFA/mol of BSA) and the number of observed binding sites decreased with increasing chain length, from 4–5 for C20:0, 3–4 for C22:0, and 2 for C24:0; we validated our previous conclusion that BSA has only one site for C26:0 (Ho, J. K., H. Moser, Y. Kishimoto, and J. A. Hamilton. 1995. J. Clin. Invest. 96: 1455–1463). Analysis of chemical shifts suggested that the highest affinity sites for VLCFA are low affinity sites for long-chain fatty acids. In competition experiments with 13C-labeled C22:0 (3 mol/mol of BSA) and unlabeled oleic acid, C22:0 bound to BSA in the presence of up to 4 mol of oleic acid/mol of BSA, but 1 mol was shifted into a different site.Our studies suggest that albumin has adequate binding capacity for the low plasma levels of VLCFA with 20 to 26 carbons, but the protein may not be able to bind longer chain VLCFA.
Highlights
The remarkable binding properties of serum albumin have been investigated extensively, but little is known about an important class of fatty acids, the very longchain saturated fatty acids (VLCFA; Ͼ18 carbons)
Spectra of oleic acid/bovine serum albumin (BSA) complexes have been analyzed in detail at a lower magnetic field (4.73 T) [10], new spectra were attained at a higher field (11.75 T) with enhanced resolution for comparison with spectra of VLCFA
4 mol oleic acid/ BSA, 13C-NMR spectra show a continuous increase in the total fatty acid carboxyl signal up to a ratio of 8 to 10 mol, at which fatty acids begins to precipitate as an acid-soap [11, 12]
Summary
The remarkable binding properties of serum albumin have been investigated extensively, but little is known about an important class of fatty acids, the very longchain saturated fatty acids (VLCFA; Ͼ18 carbons). Because very long-chain saturated fatty acids (VLCFA; acyl chain Ͼ18 carbons) are so insoluble in water, both the delivery of fatty acids to albumin and the measurement of the unbound aqueous pool of fatty acids are difficult, which hampers classical binding studies [6]. Our laboratory developed an NMR spectroscopic approach to investigate the binding interactions of fatty acids with various lipid-binding proteins, such as serum albumin and intracellular fatty acid-binding proteins [7,8,9,10]. This approach uses [13C]carboxyl-enriched fatty acids to enhance the sensitivity of 13C-NMR spectroscopy to detect subtle differences in the microenvironment of the carboxyl upon binding.
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