Biology Reference
In-Depth Information
aqueous sub-phase between two Teflon barriers, one or both of which can move parallel to
the side walls of the trough. The barriers are in constant contact with the top of the subphase.
After the organic solvent has evaporated, the barrier(s) are moved and the monolayer is
compressed. Barrier movement, and hence the size of the monolayer. is accurately controlled
by computer. The temperature of the water subphase is also carefully controlled. During the
compression, the surface tension of the monolayer is continuously monitored by use of a
Wilhelmy plate attached to an electronic linear-displacement sensor, or electrobalance
(discussed in Chapter 3). Monolayer surface pressure (
p
) is calculated by subtracting the
surface tension of the subphase with the floating monolayer (
g
) from the surface tension of
the pure subphase with no monolayer (
g
o
).
g
o
for water, the normal subphase, is a constant
at a single temperature (e.g.
73 mN/m at 20
C)
[10]
.
z
p ¼ g
0
g
Surface pressure varies with the molecular area of the compressed monolayer. Surface
tension measurements are exquisitely sensitive to any contamination, as contaminants will
often accumulate at the water/air interface where they compete with the monolayer lipids.
Since even 1 ppm contaminant can radically change monolayer behavior, maximal cleanli-
ness and purity of components must be observed. Experiments are often run in a clean
room to prevent airborne contaminants and on a vibration-free table. Dilute solutions of
the membrane lipid to be tested (~1mg/ml) are made in an organic carrying solvent that
must dissolve the amphipathic lipids while also being volatile. Examples of these solvents
currently in use include hexane/2-propanol (3:2) or ethanol/hexane (5:95). The lipids rapidly
spread over the clean aqueous interface with their polar head groups in the water and their
hydrophobic tails extended into the air. After 4
e
5 minutes to allow for the carrying solvent to
dissipate, the compression is begun.
Figure 11.2
shows pressure-area (
P
-A) isotherms for a simple, saturated fatty acid (e.g. pal-
mitic or stearic acid) on the left and a heterochain phospholipid (e.g. 18:0, 18:1 PC) on the right.
Note that the phospholipid isotherm is far more complicated than that observed for the simple
fatty acid
[11]
. At the lowest possible lipid densities, the lipids are not touching and the mono-
layer exhibits a quasi two-dimensional gas state (G). At ~78
˚
2
/molecule, depending on the
phospholipid, the lipids come into contact with one another and enter the liquid-expanded
(LE) phase. Note the LE phase is missing for un-esterified, saturated chain fatty acids. In the
LE phase, lipids are in contact but without molecular order. At ~5 mN/m a transition occurs
from the LE phase to the liquid-condensed (LC) phase that exhibits order (LE to LC). Further
compression will result in the solid (S) phase and eventually lead to collapse of the monolayer
(Collapse Point), where the lipids exhibit maximal possible density. The sequence of states that
a phospholipid monolayer goes through upon compression is therefore:
Collapse Point
It is estimated that the lateral pressure of a biological membrane is ~30
e
35 mN/m, consid-
erably less than the Collapse Point, but substantially greater than the LE phase.
Figure 11.3
shows
G
G-LE
LE
LE
=
LC
LC
S
/
/
/
/
/
/
-A curves for lipids of different compressibilities
[12]
. Curve 'a' is the
highly incompressible 16:0,16:0 PC which remains in the solid-like phase throughout the
compression; curve 'b' is compressible 22:6,22:6 PC which is similar to the curve for
14:0,14:0 PE discussed in
Figure 11.2
; and curve 'c' is mixed chain egg PC which remains
P
