The kinetics of the interaction of long-chain fatty acids (referred to as fatty acids) with albumin is critical to understanding the role of albumin in fatty acid transport. In this study we have determined the kinetics of fatty acid dissociation from BSA and the BSA-related fatty acid probe BSA-HCA (BSA labelled with 7-hydroxycoumarin-4-acetic acid) by stopped-flow methods. Fatty acid—albumin complexes of a range of natural fatty acid types and albumin molecules (donors) were mixed with three fatty acid-binding acceptor proteins. Dissociation of fatty acids from the donor was monitored by either the time course of donor fluorescence/absorbance or the time course of acceptor fluorescence. The results of these measurements indicate that fatty acid dissociation from BSA as well as BSA-HCA is well described by a single exponential function over the entire range of fatty acid/albumin molar ratios used in these measurements, from 0.5:1 to 6:1. The observed rate constants (kobs) for the dissociation of each fatty acid type reveal little or no dependence on the initial fatty acid/albumin ratio. However, dissociation rates were dependent upon the type of fatty acid. In the case of native BSA with an initial fatty acid/BSA molar ratio of 3:1, the order of kobs values was stearic acid (1.5s−1)<oleic acid<palmitic acid≅linoleic acid<arachidonic acid (8s−1) at 37°C. The corresponding values for BSA-HCA were about half the values for BSA. The results of this study show that the rate of fatty acid dissociation from native BSA is more than 10-fold faster than reported previously and that the off-rate constants for the five primary fatty acid-binding sites differ by less than a factor of 2. We conclude that for reported rates of fatty acid transport across cell membranes, dissociation of fatty acids from the fatty acid—BSA complexes used in the transport studies should not be rate-limiting.
- fatty acid-binding protein
- fatty acid probe
- fluorescence recording
Abbreviations used: BSA-HCA, BSA—7-hydroxycoumarin-4-acetic acid complex; FABP, fatty acid-binding protein; L-FABP, liver FABP; ADIFAB2, Leu72→Ala-modified rat intestinal FABP labelled with acrylodan; HSA, human serum albumin; kobs, observed rate constant; kon, koff, association and dissociation rate constants; ν, average number of fatty acid molecules bound/molecule of albumin; OA, oleic acid.
- The Biochemical Society, London ©2002