Open AccessIsolation, Purification and Physico‐Chemical Characteristic of Shigella sonnei Phase I Free Endotoxin
Author(s) -
Romanowska E.,
Pelczarska A.,
Wnuk W.,
Mulczyk M.,
Godzińska H.,
ŠLopek S.
Publication year - 1970
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1970.tb00870.x
Subject(s) - chromatography , chemistry , sephadex , shigella sonnei , ultracentrifuge , partial specific volume , size exclusion chromatography , heptose , biochemistry , escherichia coli , shigella , mutant , gene , enzyme
The specific extracellular material with endotoxic properties (free endotoxin) was isolated from liquid culture of Shigella sonnei phase I. Bio‐Gel P‐300 filtration and subsequent ultracentrifugation at 10500μg was used to purify and separate free endotoxin into two fractions: the sediment and the supernatant. The sediment fraction was identified as lipopolysaccharide‐protein complex of the molecular weight of several millions, containing the same constituents as cell lipopolysaccharide of Shigella sonnei phase I, prepared by phenol–water extraction. This substance was a better endotoxin than cell lipopolysaccharide; it showed strong toxicity (L.D. 50 = 15 μg), pyrogenicity and elicited local and general Shwartzman reaction. The supernatant fraction represents phase I specific substance practically free of heptose, 2‐keto‐3‐deoxyoctonic acid and phosphorus. It contained glucose, galactose and glucosamine as does phase I lipopolysaccharide, and also galactosamine and small amounts of mannose. Phase I specific substance was very active serologically and revealed very weak endotoxic properties (L.D. 50 = 1800 μg). The homogeneity of the supernatant fraction was proved in sedimentation experiments, free electrophoresis and rechromatography on Bio‐Gel P‐300 and Sephadex G‐200 columns. The examination of hydrodynamic properties (sedimentation coefficient, limiting viscosity number, diffusion constant and partial specific volume) of the supernatant fraction showed that this substance has a linear structure with molecular weight 90800, and diameter and length of the molecule of about 15 Å and 800 Å, respectively.