Isolation of two protein-free and chemically different lipopolysaccharides from Bordetella pertussis phenol-extracted endotoxin.

Endotoxin prepared from several Bordetella pertussis strains in both immunological phases I and IV gave two lipopolysaccharide peaks (LPS-I and LPS-II) when analyzed on hydroxylapatite columns in a phosphate buffer containing 0.1% sodium dodecyl sulfate; these lipopolysaccharides, present in the ratio of 2:3, are true endotoxins by both chemical and biological criteria. Endotoxins isolated from Escherichia coli, Salmonella typhimurium, and Shigella flexneri gave single lipopolysaccharide peaks when analyzed by the same procedure. Upon hydrolysis with acetic acid (pH 3.4) at 100 degrees C for 1 h, LPS-I released a polysaccharide (PS-I); the linkage broken was that of the glycosidic bond of a non-phosphorylated 3-deoxy-oct-2-ulosonic acid. Treatment with 0.25 M mineral acid at 100 degrees C for 30 min was required to free the polysaccharide moiety (PS-II) of LPS-II, the linkage broken being the glycosidic bond of a phosphorylated 3-deoxy-oct-2-ulosonic acid. Chemical and physical differences of the polysaccharide moieties PS-I and PS-II present in LPS-I and LPS-II have been described previously (25). By using the technique of 125I labeling, it was shown that the totality of labeled proteins present in the endotoxin extracted from Bordetella pertussis by the phenol-water procedure could be separated from the lipopolysaccharide by column chromatography on hydroxylapatite; it follows that these proteins are not linked by covalent bonds to the lipopolysaccharide.