A practical guide to structural analysis of carbohydrates

Delipidation of lipopolysaccharides with dilute acetic acid

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Structural analysis
 Lipopolysaccharides (LPS) are found in the cell wall of Gram-negative bacteria i.e. those that do retain the gram-stain. Given the general formula for enterobacterial lipopolysaccharides with an O-polysaccharide, a core and a lipid A, treatment with dilute acetic acid cleaves the ketosidic linkages of the Kdo residues.

The specific details of the O-polysaccharide, the core and the lipid A varies substantially. If the O-polysaccharide is present, then the bacteria are called smooth, if it is absent they are then called rough. The repeating units, shown in brackets above, are distributed over a range say 10-40 can be shown by sodium-dodecyl sulphate polyacrylamide gel electrophoreses (SDS-PAGE) where a ladder pattern is seen. A phosphate group at position 4 of the branch point Kdo often results in an elimination reaction and "anhydro-Kdo" is formed i.e. a Kdo molecule that formally has lost water. The result of the delipidation is thus a free O-polysaccharide linked to a modified core (with only one Kdo) and free lipid A. The delipidation is also sometimes performed in detergent when there are real solubility problems or when heterogeneous product mixtures are obtained.

Reagents

  • Aqueous 1% acetic acid
  • Diethyl ether or hexane

Procedure

  1. Suspend the LPS in water in a conical NS19 flask or suitable vessel. Sonicate until lumps of LPS (if present) are gone. Add acetic acid to a concentration of 1%.
  2. Keep the flask at 100°C for 1h.
  3. Cool the reaction mixture, transfer to centrifuge tubes and spin down the lipid pellet at 5000 rpm for 10 minutes, carefully remove and keep the supernatant. Resuspend the pellet with 3 mL of water, centrifuge, remove the supernatant and pool with the first. If no precipitate is formed extract the reaction mixture with diethyl ether or hexane using half the volume of the reaction mixture. Repeat once. Freeze dry.
  4. Dissolve in water or buffer and purify by gel filtration chromatography.

Comments

  1. The "solution" will always be cloudy due to the content of lipid in the LPS.
  2. Normally, but not always, the released lipid precipitates.
  3. The precipitate or diethyl ether/hexane extract may be kept and analysed by e.g. MS. The LPS pellet is extracted with chloroform/methanol/water (12:6:1).
  4. The purification is necessary as the reaction mixture often contains unhydrolysed LPS, a mixture of polysaccharides and oligosaccharides and salt.
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