The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins.
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.
Davidson
W Sean
WS
Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507, USA. sean.davidson@uc.edu
Ghering
Amy B
AB
Beish
Lauren
L
Tubb
Matthew R
MR
Hui
David Y
DY
Pearson
Kevin
K
D000465:Algorithms; D016632:Apolipoprotein A-I / Q000235:genetics / Q000378:metabolism; D016633:Apolipoprotein A-II / Q000378:metabolism; D001053:Apolipoproteins / Q000737:chemistry / Q000378:metabolism*; D001054:Apolipoproteins A / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001565:Benzoates / Q000737:chemistry; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods; D003619:Dansyl Compounds / Q000737:chemistry; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011804:Quinolines / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012235:Rhodamines / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001053:Apolipoproteins; D001054:Apolipoproteins A; D001426:Bacterial Proteins; D001565:Benzoates; D003619:Dansyl Compounds; D008081:Liposomes; C053685:N-(lissamine rhodamine B sulfonyl)-1,2-dioleoylphosphatidylethanolamine; C095271:N-biotinyl-dipalmitoylphosphatidylethanolamine; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011804:Quinolines; D011994:Recombinant Proteins; D012235:Rhodamines; C022205:apolipoprotein A-IV; C049538:streptavidin-agarose; C069617:3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde; C024284:dansyl phosphatidylethanolamine; D001710:Biotin; D012685:Sepharose; C028694:1-palmitoyl-2-oleoylphosphatidylcholine