Electrochemistry of Biomimetic Membranes - Applications of Electrochemistry and Nanotechnology in Biology and Medicine - page 217

pumps protons from the matrix to the intermembrane space, where
it interacts with ferro-cytochrome c . In the absence of ferro-
cytochrome c , the square-wave voltammogram of the tBLM incor-
porating COX shows a reduction peak due to the electroreduction
of protons on the gold surface at -0.70 V, as usual; increasing ad-
ditions of ferro-cytochrome c cause a progressive decrease of this
peak; this decrease was ascribed to a decrease in the proton con-
centration within the thiopeptide spacer, due to proton pumping
from the spacer to the aqueous solution. This direction of the pro-
ton flux was explained by the fact that ferro-cytocrome c can only
interact with the COX protein molecules that turn their intermem-
brane-space side toward the aqueous phase, which is also the di-
rection of COX proton pumping in biomembranes. The addition of
cyanide, which is known to inhibit proton transport by binding to
the binuclear center of COX, eliminates the effect of ferro-
cytochrome c . As already stated, thiolipopeptide-based tBLMs
have a differential capacitance much higher and a resistance much
lower than those of conventional BLMs, thus denoting poor elec-
trical properties and an appreciable field-assisted permeability to
protons. The lipid monolayer on top of the thiolipopeptide mono-
layer shows no detectable mobility. This was demonstrated by
fusing lipid vesicles labeled with a fluorescent probe, photobleach-
ing a spot of the resulting tBLM with a laser beam and monitoring
any fluorescence recovery after photobleaching (FRAP) in this
spot; no recovery was observed after ten minutes.
The above conclusions as to proton pump incorporation in
thiolipopeptide-based tBLMs should be examined in light of the
observation that vesicles have a low propensity to fuse on the hy-
drophobic surface exposed to the aqueous solution by a thiolipid
monolayer, especially if they incorporate an integral protein; ra-
ther, they are adsorbed or partially fused (cf. Section IV.2). Incor-
poration of the above proton pumps from their solutions in deter-
gent may easily take place in the membrane of adsorbed or partial-
ly fused vesicles, since the vesicular membrane is clearly inter-
posed between two aqueous phases. An analogous situation may
be envisaged with proteoliposomes that are adsorbed or partially
fused on the thiolipid monolayer from their suspending solution. In
this respect, the functional activity of the above proton pumps
might well be successfully verified even with vesicles or prote-
oliposomes adsorbed and/or partially fused on a thiolipid mono-