Rapid preparation of samples for compositional sugar analysis of the "degraded polysaccharide" fraction of lipopolysaccharides from Vibrio cholerae.

Abstract

A simple and rapid method was devised for direct isolation and fractionation of the "degraded polysaccharide" (DPS) fraction of O-antigenic (or endotoxic) lipopolysaccharides (LPS) directly from heat-killed Vibrio cholerae (O1 and non-O1) cells without separating the LPS. Neither phenol-water extraction nor ultracentrifuge is needed in this method. V. cholerae NIH 41 was used as standard. The cells (3-5 g wet weight) were heated in 5% acetic acid at 100 C for 1.5 hr. The acetic acid extract obtained as the supernatant by centrifugation was evaporated to dryness in vacuo, and the resultant residue was dissolved in 10 ml of distilled water. The solution was mixed with 2 volumes of acetone, and the supernatant obtained by centrifugation was mixed with 5 volumes of acetone and centrifuged. Fraction Sed. II was recovered as the precipitate, while the supernatant was evaporated to dryness in vacuo, yielding fraction Sup. III. Sed. II had a sugar composition that was identical, at least qualitatively, to that of DPS isolated from LPS of the corresponding strain except for the absence of a fructose component in the case of V. cholerae NIH 41, while instead Sup. III from V. cholerae NIH 41 contained fructose.