Stopped-flow kinetic analysis of long-chain fatty acid dissociation from bovine serum albumin.

Erland J F Demant, Gary V Richieri, Alan M Kleinfeld

Index: Biochem. J. 363(Pt 3) , 809-15, (2002)

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Abstract

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 (k(obs)) 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 k(obs) values was stearic acid (1.5 s(-1)) < oleic acid < palmitic acid congruent with linoleic acid