[Shigella endotoxin protein--its isolation and physicochemical characteristics]

The scheme of the isolation of endotoxic protein from S. sonnei 9090 is presented. The isolation procedure includes the 10-minute hot (at 68 degrees C) extraction of protein from endotoxin with 45% aqueous phenol, the precipitation of protein from phenolic extract with 9.5 volumes of 95% ethanol, the purification of protein from lipid material and pigments by multiple extraction with the mixture of chloroform and ethanol in the proportion 2:1 by volume. The yield of protein obtained with the use of this isolation scheme is about 3% of the initial endotoxin preparation. Protein preparations obtained in accordance with this scheme contain 92-95% of protein (determined by Lowry's method), 2.3-3.0% of saccharides (determined by the phenol-sulfate method) and 0.02% of hexose amine, its presence indicating that the preparations contain lipid A (or its fragments) which is firmly bound with endotoxic protein and cannot be extracted with chloroform. As shown in the passive hemagglutination inhibition test, the content of endotoxin in the preparations is less than 0.003%. Out of 7-11 bands revealed by electrophoresis in 15% polyacrylamide gel in the presence of sodium dodecyl sulfate, 3 main bands have molecular weights of 43, 38 and 18 KD. Three antigens differing in their electrophoretic mobility and diffusion rate in 1% agarose gel can be detected in the preparations by the method of immunoelectrophoresis with the use of antisera to both endotoxin and endotoxic protein.