Phosphatidylserine thin-layer chromatography

The ethanol-chloroform-water (5 2 1, v/v) eluate is evaporated to dryness in vacuo and the residue is dissolved in chloroform-methanol (2 1, v/v). This fraction can be analyzed for total P, and its contents can be evaluated by thin-layer chromatography on silica gel H (250 pm) plates in a solvent system of chloroform-methanol-ammonium hydroxide (28% 65 35 7, v/v). Two ninhydrin-positive spots will be found one is at Rf 0.50, which is phospha-tidylcthanolamine the other is at Rf 0.20, which is phosphatidylserine. These findings then make it possible to isolate phosphatidylserine by preparative thin-layer chromatography. 

Phospholipase A2 Action. Incubation of phosphatidylserine with phospholipase A2 obtained from Crotalus adamanteus or Naja Naja snake venom will show that the serine-containing phosphoglyceride was smoothly and completely converted to a lysophosphatidylserine with liberation of 1 mol of fatty acid per mole of lipid P. The experimental procedure was the same as the one described before in this and in the previous chapter. The products of the reaction can be recovered by thin-layer chromatography on Whatman K6 plates in a solvent system of chloroform-acetone-methanol-acetic acid-water (4.5 2 1 1.3 0.5, v/v). 

Incubation of this Na-K ATPase preparation with phosphatidylserine decarboxylase leads to complete conversion of the 31 phosphatidyl serine molecules present per molecule of Na-K ATPase, again without significant reduction in Na-K ATPase activity. In this case the sensitivity of the phosphatidylserine assay (thin layer chromatography and amino acid analysis) is such that it can be stated that less than one phosphatidylserine molecule per molecule of enz3rme is left. 

Figure 4. Analysis of the different polyphosphoinositides extracted from P-labelled cells. (A) Schematic representation of the expected separation of a mixture of P-labelled phospholipids by thin layer chromatography (TLC). Plates are silica gel 60 and the solvent for phosphoinositide separation is a mixture of CHCI3, CH3COCH3, CH3OH, CH3COOH and H O (80 30 26 24 14, v/v). MP, major phospholipids (phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine). (B) Typical high-performance liquid chromatography profile showing the separation of the various phosphoinositides from a mixture of P-labelled phosphoinositides. A specific gradient must be used to separate PtdIns(4)P and PtdIns(5)P (Rameh et ai, 1997 Niebhur et al., 2002).

Essentially three approaches can be used in the isolation of phosphatidylserine from a total lipid sample. These include thin-layer silica gel chromatography, aluminum oxide chromatography, and high-performance (high-pressure) liquid chromatography. The merits of these techniques are discussed as follows. 

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