Affinity precipitation of proteins by surfactant-solubilized, ligand-modified phospholipids.
The use of ligand-modified phospholipids solubilized in aqueous solution by nonionic surfactant for affinity precipitation of proteins is described. Avidin was precipitated by contact with solutions in which dimyristoylphosphatidylethanolamine (DMPE) functionalized with biotin (DMPE-B) was solubilized in octaethylene glycol mono-n-dodecyl ether (C12E8) solutions. The nonionic surfactant solubilizes the phospholipid in micelles above its critical micelle concentration (CMC) and in small submicellar aggregates below this concentration. At C12E8 concentrations significantly exceeding its CMC, determined to be about 100 microM, precipitation of avidin by solubilized DMPE-B is not observed. In this regime, binding of protein by DMPE-B was monitored by a hyperchroic shift in the protein's UV-visible spectrum at 231.5 nm. The data were analyzed using a model that considers the four binding sites on the protein to be independent and identical in binding strength for DMPE-B. Below the CMC of C12E8, precipitation is observed and is monitored by increasing turbidity of the solution. The kinetics of precipitation and the aggregate size measured by quasielastic light scattering were analyzed using Smoluchowski kinetics and the Mie scattering theory. These results help establish more completely the factors that influence the precipitation of proteins by ligand-modified phospholipids, and they are helpful in specifying conditions for the precipitation of other proteins.
Powers
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DD
Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7905.
Willard
B L
BL
Carbonell
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RG
Kilpatrick
P K
PK
D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D011232:Chemical Precipitation; D007700:Kinetics; D008024:Ligands; D008027:Light; D008823:Micelles; D008956:Models, Chemical; D010714:Phosphatidylethanolamines; D010743:Phospholipids / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D012542:Scattering, Radiation; D012995:Solubility; D013501:Surface-Active Agents / Q000737:chemistry*
D008024:Ligands; D008823:Micelles; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011506:Proteins; D013501:Surface-Active Agents; D001360:Avidin; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D001710:Biotin; C018525:1,2-dimyristoylphosphatidylethanolamine