TY - JOUR
T1 - The biotin-capture lipid affinity assay
T2 - A rapid method for determining lipid binding parameters for apolipoproteins
AU - Davidson, W. Sean
AU - Ghering, Amy B.
AU - Beish, Lauren
AU - Tubb, Matthew R.
AU - Hui, David Y.
AU - Pearson, Kevin
PY - 2006/2
Y1 - 2006/2
N2 - 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.jlr 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.
AB - 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.jlr 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.
KW - Biotin/streptavidin
KW - Fluorescence
KW - Lipid binding assay
KW - Small unilamellar vesicle
UR - http://www.scopus.com/inward/record.url?scp=33244475771&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33244475771&partnerID=8YFLogxK
U2 - 10.1194/jlr.D500034-JLR200
DO - 10.1194/jlr.D500034-JLR200
M3 - Article
C2 - 16267343
AN - SCOPUS:33244475771
SN - 0022-2275
VL - 47
SP - 440
EP - 449
JO - Journal of Lipid Research
JF - Journal of Lipid Research
IS - 2
ER -