Isolation and Characterization of Lipopolysaccharides - Bacterial Pathogenesis: Methods and Protocols - page 3

The carbohydrate portion of LPS consists of a relatively conserved core region
linked to a series of repeating four to six sugar units (the O-antigen). These units
can form polysaccharides with Mr in excess of 65 kDa (3,4) . Lipooligosac-
charides (LOSs) are analogous to LPSs and have carbohydrate components
that are smaller (3200-7200 kDa) than those of LPS and tend to be more
intricately branched than the LPS carbohydrates (5) . These structures can
be isolated from Haemophilus influenzae , Neisseria gonorrhoeae , Neisseria
meningitidis , Haemophilus ducreyi , Branhamella catarrhalis , and Bordetella
pertussis .
The purpose of this chapter is to describe methods for the isolation and
analysis of LPSs and LOSs. Various methods for the isolation of these glycol-
ipids have evolved since the 1930s. These include extraction with trichloracetic
acid (6) , extraction with ether (7) , extraction with water (8) , extraction with
EDTA (9) , extraction with pyridine (10) , extraction with phenol (11) , and
extraction after solubilization with sodium docecyl sulfate (SDS) (12) .
Two procedures can suffice for the isolation of LPS preparations of high
purity. These are the modified phenol-water method (13) and a modification
of the proteinase K method and phenol extraction, which allows extraction of
a LPS preparation free of contaminating lipoproteins (14) .
Because it is effective in removing contaminating proteins, the latter
method is useful for studying biological interactions with LPS. The phenol-
water technique, which was first described by Westphal and Jann (11) , takes
advantage of the amphopathic nature of the LPS and the solubility of the
majority of bacterial proteins in phenol. Polysaccharides, mucopolysaccha-
rides, LPSs with O-side chains, and nucleic acids are usually soluble in
aqueous solutions and insoluble in phenol. Phenol is a weak acid; its disso-
ciation constant at 18 °C in water is 1.2 × 10 − 10 . Mixtures of phenol

and water have a high dielectric constant. These facts form the basis of
a method of partition of proteins and polysaccharides and/or nucleic acids
between phenol and water. Minor modifications have been made to the
basic protocol described by Westphal et al. (15) and by Johnson and Perry
(16) . These changes result in LPS and LOS preparations that have less
contamination with nucleic acids and produce somewhat greater yields. One
major limitation of the phenol-water extraction method is that LPS with
truncated polysaccharide components or the more hydrophobic, shorter chain
LOSs frequently partition into the phenol phase, as Erwin and co-workers
(17) have isolated the LOS of H.

∼

influenzae aegyptius from the phenol

phase.
The combination of proteinase K digestion of bacterial proteins followed by
nuclease digestion and phenol water extraction results in an LPS preparation
of very high quality, free of contaminating proteins and nucleic acids.