Biology Reference
In-Depth Information
by changing the dialysis or gel filtration rate. Effect of detergents on vesicle size can be
dramatic
[47]
. For example:
Detergent
Vesicle size
Sodium Cholate
SUV (small)
n-octyl (or hexyl or heptyl)
b
-D-glucopyranoside
LUV (medium)
POE4 (lauryl dimethylamine oxide)
GUV (very large)
Unfortunately it is very difficult to remove all of the detergent from new liposomes
prepared by dialysis or gel filtration. This is particularly true for detergents with a low
CMC (e.g. Triton X-100). Another detergent-removal process, extraction using hydrophobic
adsorption onto non-polar polystyrene beads
[48]
, has proven to be very effective and has
become commonly used in unilamellar vesicle preparations. The best known polystyrene
product is Bio Beads
from Bio-Rad. Polystyrene beads can be used to clean up unilamellar
vesicles made by any detergent process or can be used in lieu of dialysis or gel filtration. Poly-
styrene beads are responsible for major advances in membrane integral protein reconstitu-
tions into proteoliposomes discussed below.
LUVs: Dilution Methods
LUVs can be made by variations of methods that are all based on a similar theme
rapid
dilution of a concentrated membrane lipid solution. By these methods membrane polar lipids
are dissolved in a small volume of an organic solvent. Solvents include ethanol, ether, Freon
(CHFCl
2
) or a detergent-in-water solution. The concentrated membrane lipid solution is
injected into a large excess of a rapidly mixing aqueous bathing solution. The organic solvent
or detergent is instantly dispersed into a large excess of the bathing solution as the water-
insoluble membrane polar lipids separate into LUVs. Ethanol works well as the lipid solvent
since it is infinitely miscible in water. Another method, known as 'ether vaporization', is
demonstrated in
Figure 13.17 [49]
. While ether is totally insoluble in water, it does have
a high vapor pressure and can readily be removed from water by vacuum. The membrane
lipids are dissolved at high concentration in ether and slowly injected into an aqueous
bath that is both warmed and under vacuum. The ether is instantly vaporized and pumped
away while the membrane lipids are left behind as LUVs. The Freon method is similar to the
'ether vaporization' method. Membrane lipids are dissolved at high concentration in Freon
and slowly injected into an aqueous bath maintained at 37
C. Since Freon boils at ~9
C, it
is instantly vaporized leaving behind LUVs. Finally, membrane lipids, dissolved in an
aqueous detergent solution, can be injected into a large aqueous bath where the water-soluble
detergent is rapidly dispersed leaving LUVs. The detergent dilution method has a very
important advantage over the ethanol, ether vaporization and Freon methods. Functional
integral proteins, isolated in detergent/membrane lipid micelles, can be incorporated into
LUVs as they form, a process known as 'membrane reconstitution'.
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