Isolation and characterization of Shigella shigae cytotoxin

Shigella shigae cytotoxin was isolated from pressure-dialyzed culture medium and from a 26-year-old sample of partially purified toxin. The toxin was adsorbed to a column of acid-treated chitin at low salt concentration and eluted with 1 M NaCl. The partially purified toxin was labeled with 125 I and resubmitted to chromatography on acid-treated chitin. The labeled material eluted with 1 M NaCl was mixed with unlabeled rabbit hemoglobin as a carrier and the toxin was further purified by chromatography on a DE52 column and by sucrose gradient centrifugation. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the pure Shigella toxin migrated as two bands corresponding to molecular weights of 30,500 and of about 11,000. The intact toxin may consist of one heavy chain and four to five copies of the light chain. In isoelectric focusing experiments, Shigella toxin was recovered from a broad zone between pH 5.8 and pH 7.5. This appears to be due to charge heterogeneities both in the large and the small chain. Most cell lines tested were completely resistant even to high concentrations of Shigella toxin. Vero cells and one strain of HeLa cells were very sensitive, 2.5 pg/ml of pure toxin induced 50% inhibition of protein synthesis overnight in HeLa cells.