Molecular Analysis of Lipid Macroamphiphiles by Hydrophobic Interaction Chromatography, Exemplified with Lipoteichoic Acids

Abstract

Analyzed were (I) the oligoglucosyl-, alanyl-substituted poly(glycerophosphate) lipoteichoic acid of Enterococcus hirae containing Glc(α1-2)Glc(α1-3)acyl 2-Gro and a phosphatidyl derivative thereof as lipid anchor, (II) the poly(digalactosyl, galactosylglycerophosphate) lipoteichoic acid of Lactococcus garvieae containing the same glycolipid and an acyl derivative of it, and (III) the N-acetylglucosaminyl-, alanyl-substituted poly(glycerophosphate) lipoteichoic acid of Staphylococcus aureus with Glc(β1-6)Glc(β1-3)acyl 2Gro as the sole lipid moiety. Hydrophobic interaction chromatography on octyl-Sepharose separated lipoteichoic acids I and II into two peaks according to the number of fatty acids. Within each peak further fractionation occurred in the order of decreasing length of the hydrophilic chain. A similar fractionation was observed within the single peak of lipoteichoic acid III. With lipoteichoic acid I and III the extent of glycosylation decreased with decreasing length of the hydrophilic chain whereas the content of alanine ester remained either constant or increased. Variations in the oligosaccharide pattern of lipoteichoic acid I and in the fatty acid composition of all lipoteichoic acids could also be observed. Collectively, the data provide for the first time a detailed picture of the complex polydispersity of lipoteichoic acids comprising the number of fatty acids, the length of the hydrophilic chain, the kind and extent of chain substitution, and the fatty acid composition. The procedure will also be applicable to the molecular analysis of lipopolysaccharides and bacterial lipoglycans. Moreover, the results are of physicochemical interest because they demonstrate for lipid macroamphiphiles an inverse relationship between hydrophobicity and the size of the hydrophilic headgroup.