Experiment 2:


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.


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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