Affinity Precipitation of
Proteins by Surfactant Solubilized, Ligand-Modified Phospholipids
Daniel D. Powers, Betsy L. Willard, Ruben G. Carbonell, and Peter K. Kilpatrick
Department of Chemical
Engineering,
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 phospholipids in micelles above its critical micelle concentration (CMC) and
in small submicellar aggregates below this concentration.
At concentrations significantly exceeding its CMC, determined to be about 100 μM, 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 a t 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.