Phenol-water extraction

I Delipidation I Hot phenol-water extraction I Digestion with RNase-DNase... 

Hot phenol-water extraction Digestion with RNase-DNase Hot phenol-water extraction... 

The vaccine formulation comprises a mixture of the outer-membrane lipopolysaccharides from bacteria of the seven different serotypes. The lipopolysaccharides may be obtained from cultures of each of the seven types by the standard procedure of Westphal (11), involving phenol—water extraction of the cells. However, for large-scale purposes, the older procedure of Boivin, (12) involving extraction of the cells with trichloroacetic acid, was... 

The lipid material precipitated upon mild acid treatment of the Boivin-extracted lipopolysaccharide is here termed a lipoidal precipitate. The fatty-acid profile (Table IV) of hydrolyzates of this material shows little variation between the seven lmmunotype strains. If the original lipopolysaccharides are first treated by phenol—water extraction, and the resultant materials then subjected to hydrolysis to release the lipid, the composition of the latter is significantly different it corresponds closely to the classic composition expected for lipid A. It is noteworthy that material extracted by the phenol—water (Westphal) method is rich in the Csaturated acid and in the hydroxy fatty acids having ten and twelve carbon atoms, whereas the and C g saturated acids present in the lipoidal precipitate, as prepared by the Boivin procedure, are absent or present at much lower levels in the lipid prepared by the Westphal procedure -9). It... 

As obtained by phenol—water extraction of the lipoidal precipitate. 

Because LPS molecules in different bacteria have different structures and amphi-pathic properties, there is no universal method for extraction of LPS. Several methods have been developed each favors some specific groups of LPS. For example, the phenol-water extraction method favors S-LPS extraction, but not R-LPS (Hickman and Ashwell, 1966 Kasai and Nowotny, 1967) the ether extraction method favors the extraction of R-LPS (Galanos et al., 1969). Both methods for large scale or micro-scale extractions have been developed. Agents used by different extraction methods are listed in Table 2.1. 

Since the discovery of LPS (Shear, 1941), various methods for the extraction of LPS have been developed. These include extraction with trichloroacetic acid (Ribi et al., 1961), with ether (Galanos et al., 1969), with water (Robert et al., 1967), with pyridine (Goebel et al., 1945), with phenol (Westphal and Jann, 1965), with butanol (Morrison and Leive, 1975), and with sodium dodecyl sulfate (SDS) (Darveau and Hancock, 1983). A few of these methods can chemically alter the structure of LPS (Nowotny et al., 1966 Tsang et al., 1974 Wober and Alaupovic, 1971). Among these methods, the most used are the phenol-water extraction (Westphal and Jann, 1965) and the ether extraction (Galanos et al., 1969). The former is most efficient for the extraction of S-LPS, while the latter for R-LPS. A method which could efficiently extract both S-LPS and R-LPS has also been developed (Darveau and Hancock, 1983). 

The mixture of phenol and water (45 50, v/v) can be used to extract LPS (Westphal and Jann, 1965). This mixture is a single phase above 65°C but separates into two phases below 65°C. LPS and proteins can be extracted from bacteria by this mixture above 65°C. When cooled down, phase separation occurs. The phenol phase mainly contains proteins, while the water phase contains LPS, polysaccharides, and nucleic acids. The following protocol is the most used for phenol-water extraction of LPS (Johnson and Perry, 1976). 

One major limitation of the phenol-water extraction method is that R-LPS frequently partitions into the phenol phase (Hickman and Ashwell, 1966 Kasai and... 

Since SR-LPS of H. pylori NCTC 11637 isolated from phenol-water extraction was partially water-insoluble, and could be isolated as a pellet by high-speed centrifugation, detailed studies of core OS sttucture were carried out on material obtained after treatment with aqueous acetic acid under standard conditions. Compositional analysis of the liberated OS crude preparation showed the presence of significant amounts of sugar constituents from O-chains in addition to Hep and Glc core units. Separation of the OS preparation by GPC on Bio-Gel afforded in... 

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... 

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. 

The phenol-water extraction method can contain small amounts of lipoproteins that could confound experiments designed to measure only LPS biological activity. These contaminating proteins can be removed by an alternative procedure described below, which incorporates proteinase degradation prior to... 

Occasionally, analytical studies such as western blot analysis of LPS or LOS will require preparation of same quantities of these glycolipids from multiple bacterial samples. Two methods have been described which can be used for such isolations (18,19). One relies on a modified phenol-water extraction (20), and the second utilizes SDS solubility of bacteria followed by enzymatic degradation of bacterial proteins (19). Neither preparation gives a highly purified preparation, but both enrich for LPS or LOS and can serve as adequate substitutes in acrylamide gel and western blot studies. These two methods are presented below. 

LPS may be extracted from intact cells by a variety of procedures. Treatment with chelating agents, such as EDTA, releases about one-third of the LPS. Generally LPS is obtained by a phenol-water extraction. In essence the bacterial cells are treated for Ih with 45 per cent (w/v) aqueous phenol at 68°C. On cooling the mixture separates into two phases, a lower phenol layer and an upper aqueous phase, in which the LPS is located. This treatment extracts up to 90 per cent of the LPS which can then be subjected to chemical analysis. 

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