The lipophorin molecule that we isolated
from locust hemolymph functions as a re-usable lipid
shuttle. HDLp has a density of approx. 1.08 g/ml and contains
less lipids than the loaded form of the proteins, LDLp (density
1.03 g/ml). Lipids are normally stored
as neutral glycerides, mostly triglycerides (triacylglycerol),
and in many organisms this is also the lipid that is carried through
the blood by lipoproteins. Insects, however, are special because
they transport mainly diacylglyerol. The lipophorin particles
contain also substantial amounts of other lipid classes. In this
experiment, we will determine the ratio of the different lipid
classes in the lipoprotein.
The first step is to extract the lipids from the protein, which was isolated in the preceding experiment.
In this experiment, the lipids will be separated by thin
layer chromatography, and finally we will quantitate the amount
of each lipid. This quantification is done indirectly: the individual
lipids will be eluted from the silica gel, and the amount and
composition of fatty acids determined by gas
chromatography after transmethylation.
In order to monitor any loss of lipids during the extraction and
separation steps, a known amount of unnatural lipids (the composition
is given in table 1), was added to the lipoprotein solution prior
to extraction. In insects, only even numbered fatty
acids occur; thus, we can use odd numbered fatty acids as
internal standards.
Table 1. Composition and molecular weights of the non-insect internal
standards provided.
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Class name Mr
_______________________________________________________
PL 15:0 dipentadecanoyl-phosphatidylcholine 734
MAG 17:0 monoheptadecanoin 344
DAG 15:0 1,3-dipentadecanoin 541
FFA 17:0 heptadecanoic acid 271
TAG 15:0 tripentadecanoin 878
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