Biology Reference
In-Depth Information
riddled with tiny pores that are large enough to be penetrated by a solute, but small enough
to exclude a liposome. As a result, as the solute/liposome mixture passes down the gel filtra-
tion column, the solute enters the pores and so travels a longer, more convoluted path to
reach the end of the column than does the large, excluded liposomes. Liposomes leave the
column before the non-liposome-sequestered solute. Of course, solutes initially sequestered
inside the liposomes pass through the gel and exit the column with the liposome fraction. The
solute 'sees' ~80% of the total column volume, while the liposome only 'sees' ~35% of the
volume. As an example, liposomes could be made in a 100 mM KCl buffer and passed
through the gel filtration column using a large volume of a 100 mM NaCl buffer. The lipo-
somes exiting the column would have an interior concentration of 100 mM KCl and 0 mM
NaCl, while the extra-liposomal solution would be 0 mM KCl and 100 mM NaCl. The estab-
lished trans-membrane gradients of K
þ
and Na
þ
can be made to parallel those of living cells.
Another method that can accomplish many of the same objectives as gel filtration chroma-
tography is filtration using dialysis tubing. Like gel filtration, dialysis tubing can be used to
separate large from small particles. Dialysis tubing is a thin semi-permeable sheet made of
cellulose or cellophane (a form of cellulose). The sheet has microscopic holes that allow small
solutes to pass through while retaining large particles. Dialysis tubing can be obtained of
different overall size and with different cut-off pore diameter. For example, liposomes are
made in a buffer containing a solute (A) and sealed in a dialysis tube. The tube is floated in
a large excess of buffer devoid of (A), but containing solute (B) at the same osmotic strength.
During dialysis (A) goes down its concentration gradient through the dialysis tube pores into
the bathing solution where it is readily washed away. Liposomes containing (A) cannot
squeeze through the pores and can then be removed from the dialysis tube for further exper-
imentation. Similar to the example described for gel filtration, liposomes made in 100 mM KCl
can be sealed in a dialysis tube and floated in a large excess of the same buffer but containing
100 mM NaCl in place of 100 mM KCl. Ideally, upon completion of dialysis, the tube contains
liposomes with 100 mMKCl sequestered inside and 100 mMNaCl outside. Although gel filtra-
tion and dialysis theoretically result in the same final product, dialysis is many, many times
slower. While complete dialysis may take days, gel filtration is complete in minutes. During
extended dialyses, lipids can oxidize and hydrolyze, producing products that are deleterious
to lipid bilayer structure and, in addition, the entire sample is ripe for bacterial growth.
Measurement of leakage rates from liposomes has a wide variety of applications and as
a result many methods are available. One of the most common and sensitive methods uses
14
C-glucose (or any other small, water-soluble, radio-labeled molecule) as the solute. After
the initial dialysis that produces the
14
C-glucose sequestered liposomes, the final product
is then sealed into a fresh dialysis tube and placed in a new bathing solution devoid of
14
C-glucose. As
14
C-glucose leaks out of the liposomes, it can be detected at low levels outside
the dialysis bag by a scintillation counter. Similarly, gel filtration can be used to measure
14
C-glucose leakage. Aliquots of the final
14
C-glucose-sequestered liposomes can be taken
at different times and run through a small gel filtration column. The
14
C-glucose still seques-
tered in the liposomes comes off the column first while the leaked (non-sequestered)
14
C-glucose elutes in a later fraction. The ratio of non-sequestered/sequestered
14
C-glucose
as a function of time represents the leakage rate.
Gel filtration and dialysis account for the production of most solute-sequestered lipo-
somes. However, in some specific examples the second dialysis or gel filtration steps used
