Abstract
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 microg of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.
Highlights
The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism
We highly recommend that prequantified samples of the apolipoprotein under study be used for the standard curve, as we have found that the CBQCA assay can give disparate results between different proteins
The sensitivity of the fluorescence lipid and protein detection allows the consumption of only 120 Ag of apolipoprotein for each protein being measured, reducing the amount of recombinant protein that needs to be purified for characterization
Summary
The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism. 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 more sophisticated strategies include the use of calorimetry [8,9,10,11,12], surface chemistry [13, 14], oil-drop tensiometry [15], capillary electrophoresis [16], or resonance energy transfer [17, 18] These methodologies can characterize the lipid binding reaction in great detail.
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