Interactions of myristic acid with bovine serum albumin: a 13C NMR study.

Abstract

Interactions of myristic acid with bovine serum albumin were studied by 13C NMR spectroscopy at 50.3 MHz using 90% isotopically substituted [1-13C]-, [3-13C]-, and [14-13C]myristic acids, either individually or in a combination of all three with albumin. At pH 7.4, two or more resonances of different intensities were observed for each 13C-enriched myristic acid. Carboxyl and methylene C-3 resonances corresponding to the major myristic acid environment(s) exhibited pH-dependent chemical shift changes indicative of protonation below pH 6.7; in contrast, carboxyl groups in minor environments were resistant to protonation. 13C NMR spectra obtained as a function of the molar ratio of [3-13C]- and [14-13C]myristic acid to bovine serum albumin (from 0.7 to 5.6) revealed at least two narrow resonances for each carbon at all molar ratios. Thus, bovine serum albumin binding sites for myristic acid are heterogeneous with respect to titration behavior and with respect to the local magnetic environment at both the polar and the nonpolar ends of the fatty acid. The narrow resonances observed for the methylene and methyl carbons are inconsistent with complete immobilization of the protein-bound acid molecules. Together with spin- lattice relaxation times and nuclear Overhauser enhancements, the linewidth results indicate that bound myristic acid has internal motions that are rapid compared with overall protein tumbling and that the C-3 methylene carbon is more restricted than the terminal methyl carbon.