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Originally published In Press as doi:10.1194/jlr.D500034-JLR200 on November 2, 2005

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Journal of Lipid Research, Vol. 47, 440-449, February 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Methods

The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins

W. Sean Davidson1, Amy B. Ghering, Lauren Beish, Matthew R. Tubb, David Y. Hui and Kevin Pearson

Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507

Published, JLR Papers in Press, November 2, 2005.

1 To whom correspondence should be addressed. e-mail: sean.davidson{at}uc.edu

The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism. Mutagenesis techniques have been widely used to modulate apolipoprotein lipid affinity for studying biological function, but the approach requires rapid and reliable lipid affinity assays to compare the mutants. Here, we describe a novel method that measures apolipoprotein binding to a standardized preparation of small unilamellar vesicles (SUVs) containing trace biotinylated and fluorescent phospholipids. After a 30 min incubation at various apolipoprotein concentrations, vesicle-bound protein is rapidly separated from free protein on columns of immobilized streptavidin in a 96-well microplate format. Vesicle-bound protein and lipid are eluted and measured in a fluorescence microplate reader for calculation of a dissociation constant and the maximum number of potential binding sites on the SUVs. Using human apolipoprotein A-I (apoA-I), apoA-IV, and mutants of each, we show that the assay generates binding constants that are comparable to other methods and is reproducible across time and apolipoprotein preparations. The assay is easy to perform and can measure triplicate binding parameters for up to 10 separate apolipoproteins in 3.5 h, consuming only 120 µg of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.

Supplementary key words lipid binding assay • fluorescence • small unilamellar vesicle • biotin/streptavidin

Abbreviations: apoA-I, apolipoprotein A-I; BCLA, biotin-capture lipid affinity; BT-PE, (biotinyl)-1,2-dipalmitoyl phosphatidylethanolamine; Dansyl-PE, (5-dimethylamino-1-naphthanesulfonyl)-1,2 dioleoylphosphatidylethanolamine; DMPC, dimyristoylphosphatidlylcholine; Kd, equilibrium dissociation constant; n, maximal binding of an apolipoprotein to a given lipid surface; PL, phospholipid; POPC, 1-palmitoyl,2-oleoyl phosphatidylcholine; Rhod-PE, (N-lissamine rhodamine B sulfonyl)-1,2 dioleoyl phosphatidylethanolamine; SUV, small unilamellar vesicle


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