Chemistry Reference
In-Depth Information
When alamethicin is added to a ternary vesicle system comprising PDA, phospholi-
pid, and lipopolysaccharide (LPS), the addition of polymyxin, an LPS-binding anti-
biotic, sensitizes the vesicles to alamethicin (Katz et al. 2003). Cholesterol-containing
PDA liposomes have been used to colorimetrically detect streptolysin O, a
cholesterol-dependent pore-forming toxin (Ma and Cheng 2005).
12.2.6. Membrane Active Enzymes
Enzymes that act upon membrane-bound lipids can be conveniently assayed using
PDA liposomes. Vesicles of PDA and DMPC were prepared and incubated with
phospholipase A2, an enzyme that hydrolyzes the myristoyl ester in DMPC
(Jelinek et al. 1998). The CR of the solution increases in a time-dependent manner
after addition of the enzyme. Other enzymes that used phospholipids as substrates,
such as bungaratoxin and phospholipases C and D, all exhibit similar time-dependent
increases in the CR values. These chromatic transitions arise as a result of membrane
perturbations caused by the various hydrolyzed products of the enzymatic reactions.
This assay can be used to distinguish between lipids from bacteria and archaea
(Rozner et al. 2003). In contrast to bacterial and eukaryotic membranes, archaeal
membranes contain ether linked lipids that are resistant to phospholipase mediated
hydrolysis. Thus, when liposomes containing membrane extracts from the prokaryote
E. coli and the archaea Haloferax volcanii are incubated with phospholipase A2, the
CR from the former liposomes is three times higher than that from the vesicles con-
taining lipids from H. volcanii. Other enzymes examined included sphingomyelinase
and galactosidase, using sphingomyelin and galactosyl ceramide containing vesicles,
respectively (Rozner et al. 2003).
12.2.7. Pattern Recognition
The use of pattern recognition principles in molecular recognition has had a
tremendous influence on sensor development (Albert et al. 2000; Jurs et al. 2000;
Collins and Anslyn 2007). Several reports describe the use of these principles with
both PDA sensors that detect analytes using these principles. Vesicles prepared
from tyrosine (Tyr) or tryptophan (Trp) terminated diyne lipids were used to
prepare sensors for bacterial LPSs (Rangin and Basu 2004). Exposure of the Tyr
or Trp containing liposomes to LPS from a given bacterial species under a variety
of experimental conditions (temperature, additives) afforded CR values for each of
the eight assay conditions. This panel of CR values served as a diagnostic fingerprint
for each species of LPS examined (Fig. 12.6).
Pattern recognition using calixarene receptors that bind to proteins via surface
electrostatic interaction has been used to identify a variety of proteins (Kolusheva
et al. 2006). Amphiphilic calixarenes terminated with either amino or phosphate
groups were incorporated into mixed PDA/phospholipid vesicles, which were incu-
bated with various proteins that differed in their isoelectric points (pI). As expected,
proteins with low pI values resulted in a large CR with liposomes containing cationic
calixarene receptors. Each protein was characterized by a unique DCR value, where
