HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: D500034-JLR200v1 47/2/440    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Davidson, W. S. Articles by Pearson, K. Search for Related Content PubMed PubMed Citation Articles by Davidson, W. S. Articles by Pearson, K. Social Bookmarking                      What's this?

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; K_d, 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

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. F. Oram, G. Wolfbauer, C. Tang, W. S. Davidson, and J. J. Albers An Amphipathic Helical Region of the N-terminal Barrel of Phospholipid Transfer Protein Is Critical for ABCA1-dependent Cholesterol Efflux J. Biol. Chem., April 25, 2008; 283(17): 11541 - 11549. [Abstract] [Full Text] [PDF]

				M. R. Tubb, R. A. G. D. Silva, K. J. Pearson, P. Tso, M. Liu, and W. S. Davidson Modulation of Apolipoprotein A-IV Lipid Binding by an Interaction between the N and C Termini J. Biol. Chem., September 28, 2007; 282(39): 28385 - 28394. [Abstract] [Full Text] [PDF]