Purification of lipoteichoic acid by chromatography in water-organic solvent systems

Abstract

Lipoteichoic acid (LTA), extracted from Streptococcus mutans 10449 by hot aqueous phenol, was partially purified by Sepharose 6B column chromatography in 0.01 M sodium acetate, pH 6.0, containing 0.25 M sodium chloride and 0.001 M EDTA. Nucleic acid and polysaccharide were precipitated from the LTA-containing column peak by the addition of 2 volumes of chloroform-methanol (1:5). The resulting single-phase chloroform-methanol-water (1:5:3) supernatant contained LTA and small amounts of several contaminating substances as indicated by reverse-phase high-pressure liquid chromatography and chemical analyses. LTA was purified further by DEAE-cellulose chromatography, using a concentration gradient of sodium chloride in chloroform-methanol-water (1:5:3). Two column peaks of LTA were found to contain phosphate, glycerol, glucose, and fatty acids at molar ratios of 1:1:0.11:0.10 and 1:1:0.09:0.04, respectively. The LTA polymers contained 18 and 22 repeating units of unsubstituted glycerophosphate and two glucose residues. The LTA in one column peak had two fatty acids per molecule, whereas that in the second peak contained only one. The yield of LTA was 1.68 mg per g of cell dry weight or 65 mg per g of phenol-water-extracted material. The specific activity of the LTA preparation was increased 128-fold by the purification scheme as determined by a erythrocyte-binding assay. Reverse-phase high-pressure liquid chromatography may be used for rapid separation of LTA molecules containing different numbers of acyl groups.