Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

Supported lipid membranes for reconstitution of membrane proteins
surrounded with an aqueous phase on both sides [113]. A successful streptavidin
binding experiment with a biotinylated lipid layer was performed. The same group
extended the investigations on the self-assembled amphiphilic polymer in a later
publication [114] to improve the barrier properties of the first monolayer and to
confirm that a water layer exists between the support and the lipid membrane.
In a later work a reactive polymer was chemisorbed on a functionalised glass slide
thus forming a thin polymer film on the solid support [28]. Hydrophilic amino groups
were linked to the polymer and a phospholipid bilayer was transferred to the ca. 80 Å
thick hydrated polymer film. The first layer was obtained with Langmuir-Blodgett
techniques and the second leaflet with Langmuir-Schäfer techniques. The fluidity of the
lipids was investigated with photobleaching techniques and it was found that the lipid
bilayer was fluid and stable for several days.
A hydrophilic polymer cushion with a smooth outer surface, is easily self-assembled
at the electrode surface by alternate adsorption of polycations and polyanions [ 1 15].
Such a surface was employed as a support for deposition of phospholipid multilayers
with LB-techniques and impedance spectroscopy was employed to investigate if the
polyelectrolyte was suitable as a support for an artificial cell membrane. A bilayer lipid
membrane was obtained with a membrane capacitance of ca 0.60 µF cm-2 that was
linked to the polyelectrolyte film via a calcium bridge provided the outermost layer was
negatively charged [ 116]. A similar polymer cushion was prepared on a gold electrode,
consisting of three layers of polyelectrolyte with an electroactive polycation
sandwiched between thin layers of polystyrenesulphonate. Vesicles containing
cytochrome c oxidase were fused on this surface and a biomembrane containing the
active enzyme was formed resting on the polyelectrolyte surface. The activity of the
enzyme was confirmed with amperometry in a FIA system by monitoring the transient
current from oxidation of a pulse of reduced cytochrome c at anaerobic conditions. The
fused lipid layer did not block the cytochrome c from being oxidised directly, either at
the gold electrode or via the osmium complex in the film resulting in high background
currents. However, inhibition of the enzyme with sodium azide resulted in a
temporarily decreased cytochrome c signal, which is a clear indication of an active
membrane protein. Moreover, only biomembranes with a mean thickness
corresponding to a transmembrane-containing lipid membrane, Le.: ca 60 Å resulted in
reproducible results and the enzyme kinetics estimated from a fitted Michaelis - Menten
relationship was in good agreement with literature values [117].
Another and rather amazing way to form a polymer-cushioned lipid bilayer was
recently reported and involves firstly the fusion of small unilamellar
dimyristoylphosphatidyl-choline vesicles to form an intact bilayer on a quartz substrate.
Secondly, when the cationic polyethyleneimine, PEI; is added to the solution it creeps
beneath the bilayer and forms a 40 Ångström thick soft cushion between the lipid layer
and the solid support. The process is monitored with Neutron Spectroscopy and
interestingly an attempt to fuse vesicles on a solid support already covered with the
polyelectrolyte failed [ 11 8]. In a subsequent paper it was found that if the PEI-coated
slide was allowed to dry before it was incubated with vesicles, the fusion was
successful and a continuous bilayer could be formed on top of the polyelectrolyte
[119]. Instead of building a polymer-cushioned lipid monolayer on a solid support step
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