Use of Microsphere‐Supported Phospholipid Membranes for Analysis of Protein‐Lipid Interactions

Gary E. Gilbert1

1 Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.3
DOI:  10.1002/0471142956.cy1303s34
Online Posting Date:  November, 2005
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Abstract

Many proteins bind to phospholipid membranes in order to gain or modify function. As an example, blood coagulation proteins must bind to phosphatidylserine‐containing membranes in order to gain procoagulant activity. The flow cytometry approaches described in this unit provide versatile assays for equilibrium and kinetic binding studies to measure essential membrane‐binding properties. These assays are rapid, require only small quantities of the protein of interest, and can evaluate binding interactions in complex, physiological mixtures, including blood plasma and tissue culture media.

Keywords: lipospheres; phosphatidylserine; phosphatidylcholine; factor VIII; factor V; factor IX; factor X; prothrombin; lactadherin; annexin V

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Lipospheres
  • Basic Protocol 2: Equilibrium Binding Studies
  • Basic Protocol 3: Competition Binding Studies with Phospholipid Vesicles
  • Support Protocol 1: Cleaning and Size‐Sorting of Glass Microspheres
  • Support Protocol 2: Preparation of Phospholipid Vesicles
  • Support Protocol 3: Analysis of Data: Quadratic Versus Approximate Solution
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Lipospheres

  Materials
  • Glass or silica microspheres in distilled, deionized water (see protocol 4)
  • Sonicated phospholipid membrane vesicles of interest (see protocol 5)
  • Sonicated phospholipid carrier vesicles (usually sonicated vesicles of egg yolk phosphatidylcholine; see protocol 5)
  • TBS/BSA/PLV buffer (see recipe)
  • High‐intensity bath sonicator
  • 15‐ml conical polystyrene centrifuge tubes
  • Low‐speed centrifuge with swinging‐bucket rotor

Basic Protocol 2: Equilibrium Binding Studies

  Materials
  • Lipospheres with membranes of interest (see protocol 1)
  • Control lipospheres with membranes of carrier phospholipid (e.g., phosphatidylcholine; see protocol 1)
  • Fluorophore‐labeled ligand (fluorescein maleimide‐labeled factor VIII)
  • Binding buffer (e.g., TBS/BSA/PLV; see recipe)
  • Hemacytometer
  • Flow cytometer analysis tube
  • Flow cytometer with suitable wavelength for excitation of the fluorophore used and appropriate filters for collection of the fluorescence emission

Basic Protocol 3: Competition Binding Studies with Phospholipid Vesicles

  Materials
  • Fluorophore‐labeled ligand (e.g., fluorescein‐labeled factor VIII)
  • Defined phospholipid vesicles: usually sonicated vesicles ∼20 to 30 nm in diameter, extruded vesicles ∼70 to 200 nm in diameter, or large multilamellar vesicles ∼200 to 1,000 nm in diameter (see protocol 5)
  • Lipospheres (e.g., membranes of 4% phosphatidylserine, 20% phosphatidylethanolamine, and 76% phosphatidylcholine; see protocol 1)
  • Binding buffer (e.g., TBS/BSA/PLV; see recipe)
  • Flow cytometer analysis tubes
  • Flow cytometer with suitable wavelength for excitation of the fluorophore used and appropriate filters for collection of the fluorescence emission.

Support Protocol 1: Cleaning and Size‐Sorting of Glass Microspheres

  Materials
  • 1.6‐µm (nominal diameter) glass (Duke Scientific) or solid silica (Bangs Laboratories) microspheres
  • 0.02% powdered laboratory detergent solution (e.g., Sparkleen), filtered
  • Methanol, reagent grade
  • 5% H 2O 2/5% HCl
  • 15‐ml centrifuge tube
  • Bath sonicator
  • Centrifuge
  • 3‐µm pore polycarbonate filter, 25‐mm diameter with holder (Millipore)
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Figures

Videos

Literature Cited

Literature Cited
   Bangham, A.D., Standish, M.M., and Watkins, J.C. 1965. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13:238‐252.
   Bardelle, C., Furie, B., Furie, B.C., and Gilbert, G.E. 1993. Kinetic studies of factor VIII binding to phospholipid membranes indicate a complex binding process. J. Biol. Chem. 268:8815‐8824.
   Brian, A. and McConnell. H. 1984. Allogeneic stimulation of cytotoxic T cells by supported planar membranes. Proc. Nat. Acad. Sci. U.S.A. 81:6159‐6163.
   Chen, P.S., Jr., Toribara, T.Y., and Warner, H. 1956. Microdetermination of phosphorus. Anal. Chem. 28:1756‐1758.
   Gilbert, G.E. and Arena, A.A. 1995. Phosphatidylethanolamine induces high‐affinity binding sites for factor VIII on membranes containing phosphatidyl‐L‐serine. J. Biol. Chem. 270:18500‐18505.
   Gilbert, G.E. and Arena, A.A. 1998. Unsaturated phospholipid acyl chains are required to constitute membrane binding sites for factor VIII. Biochemistry 37:13526‐13535.
   Gilbert, G.E. and Drinkwater, D. 1993. Specific membrane binding of factor VIII is mediated by O‐phospho‐L‐serine, a moiety of phosphatidylserine. Biochemistry 32:9577‐9585.
   Gilbert, G.E., Furie, B.C., and Furie, B. 1990. Binding of human factor VIII to phospholipid vesicles. J. Biol. Chem. 265:815‐822.
   Gilbert, G.E., Drinkwater, D., Barter, S., and Clouse, S.B. 1992. Specificity of phosphatidylserine‐containing membrane binding sites for factor VIII: Studies with model membranes supported by glass microspheres (lipospheres). J. Biol. Chem. 267:15861‐15868.
   Hauser, H., Pascher, I., Pearson, R.H., and Sundell, S. 1981. Preferred conformation and molecular packing of phosphatidylethanolamine and phosphatidylcholine. Biochim. Biophys. Acta 650:21‐51.
   Hope, M.J., Bally, M.B., Webb, G., and Cullis, P.R. 1985. Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812:55‐65.
   Lasic, D.D. 1997. Colloid chemistry. Liposomes within liposomes. Nature 387:26‐27.
   Lauer, S., Goldstein, B., Nolan, R.L., and Nolan, J.P. 2002. Analysis of cholera toxin‐ganglioside interactions by flow cytometry. Biochemistry 41:1742‐1751.
   Shi, J. and Gilbert, G.E. 2003. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites. Blood 101:2628‐2636.
   Shi, J., Heegaard, C.W., Rasmussen, J.T., and Gilbert, G.E. 2004. Lactadherin binds selectively to membranes containing phosphatidyl‐L‐serine and increased curvature. Biochim. Biophys. Acta 1667:82‐90.
   Stuart, M.C., Reutelingsperger, C.P., and Frederik, P.M. 1998. Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer. Cytometry 33:414‐419.
   Tamm, L. and McConnell, H. 1985. Supported phospholipid bilayers. Biophys. J. 47:105‐113.
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