出版社：American Society for Biochemistry and Molecular Biology
摘要：The lipid affinity of plasma apolipoproteins is an importantmodulator of lipoprotein metabolism. Mutagenesis techniqueshave been widely used to modulate apolipoprotein lipid affinityfor studying biological function, but the approach requiresrapid and reliable lipid affinity assays to compare the mutants.Here, we describe a novel method that measures apolipoproteinbinding 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 proteinon columns of immobilized streptavidin in a 96-well microplateformat. Vesicle-bound protein and lipid are eluted and measuredin a fluorescence microplate reader for calculation of a dissociationconstant and the maximum number of potential binding sites onthe SUVs. Using human apolipoprotein A-I (apoA-I), apoA-IV,and mutants of each, we show that the assay generates bindingconstants that are comparable to other methods and is reproducibleacross time and apolipoprotein preparations. The assay is easyto perform and can measure triplicate binding parameters forup to 10 separate apolipoproteins in 3.5 h, consuming only 120µg of apolipoprotein in total. The benefits and potentialdrawbacks 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
