Degradation of Lipopolysaccharide of Escherichia Coli by a Hot Phenol Extraction

Abstract

The extraction of lipopolysaccharides (LPS) from Escherichia coil 6234 was studied. It was found that approximately one-half of LPS was extracted with cold phenol-water without stirring (4-LPS). The remaining LPS was extracted with hot phenol-water with stirring (470-LPS). When the cells were extracted directly with hot phenol-water, all LPS was extracted in the aqueous layer (70-LPS). The three preparations were partially purified and their chemical compositions were examined. Marked differences in the chemical compositions were observed between the 4-LPS and 470-or 70-LPS. The 4-LPS was rich in fatty acids and hexosamine in lipid A moiety. The 470-and 70-LPSs showed similar chemical compositions. When the 4-LPS was treated with hot phenol-water, a partial degradation of the lipid A moiety was observed. These results were discussed with respect to the native state of LPS in the outer membrane of the envelope.Many methods have been developed for the extraction of lipopolysaccharides (LPS) from gram-negative bacteria (1, 2). One of the methods, which is widely used, is the hot phenol-water procedure yielding LPS in water phase (3,4). However, there are several reports on unexpected results. HICKMAN and ASH-WELL (5), KASAI and NOWOTNY (6), and RAFF and WHEAT (7) studied strains of Xanthomonas campestris, a heptoseless R mutant of Salmonella minnesota, and Citrobacter freundii, respectively, and found that the corresponding LPSs were obtained almost exclusively in phenol phase.WESTPHAL and JANN (3) isolated LPS in the aqueous phase by shaking bacteria in an emulsion of equal volumes of phenol and water at a low temperature (5-10°)

Production of a Monoclonal Antibody That Recognizes the Lipopolysaccharide of a Campylobacter‐Like Organism

“…4, lane 3). The high temperatures involved (65-68 C) in the isolation of LPS using the RIM procedure has some degradative effect (37,51), causing the complete elimination of the 19 kDa band as also shown in the silver-stained gel (Fig. 3, lane 3).…”

Effect of Lipid a and Fatty Acids on Transport Activities of Membrane Vesicles From Escherichia Coli

“…Lipopolysaccharide was extracted by the method essentially identical to that of OsBORN (15). The crude lipopolysaccharide was partially purified according to the method described by OKUDA et al (16), except that the Pronase digestion was omitted. The purified lipopolysaccharide was hydrolyzed with 1 % acetic acid to prepare the lipid A fraction (16).…”

“…The crude lipopolysaccharide was partially purified according to the method described by OKUDA et al (16), except that the Pronase digestion was omitted. The purified lipopolysaccharide was hydrolyzed with 1 % acetic acid to prepare the lipid A fraction (16). Lipid A was dissolved in Me2SO at a concentration of 4 mg lipid A/100 ,u1.…”

Immunochemical characterisation of Vibrio cholerae O139 O antigens and production of a diagnostic antiserum without absorption

“…This degradation may be a result of heating for 2 h at 1008C (mild hydrolysis). Indeed, the hot phenol-water technique has been shown to provide partial degradation of LPS during the isolation procedure [20,21].…”