Characterization of Shigella dysenteriae 1 (Shiga) toxin purified by anti-Shiga toxin affinity chromatography

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Abstract
S. dysenteriae 1 (Shiga) toxin was purified from whole-cell lysates by antitoxin affinity column chromatography, radioiodination and Sephacryl S-200 gel filtration of 125I-labeled affinity column eluates. Two chromatographic peaks were observed. The percentage of radioactivity in peak I samples immunoprecipitated with antitoxin ranged from 95-100%. A pool of samples from this 1st peak contained over 90% of the HeLa[human cervical carcinoma]-cell-cytotoxic units applied to the column and was enterotoxic for rabbit ileal loops and lethal for rabbits. This radiolabeled material migrated as a single cytotoxic band after nondenaturing polyacrylamide gel electrophoresis, but formed 3 bands of 33,000, 29,000 and 4000-7000 daltons after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Material estimated as 7000 daltons by Bio-Gel P-10 chromatography could be generated by treatment of S-200 peak I samples with 8 M urea. Pooled fractions from the 2nd S-200 peak were separable into several low-MW peaks on a P-10 column. One of these P-10 peaks (7000 daltons) was 27% immunoprecipitable with antitoxin. Thus 3 of the known biological activities of Shiga toxin are associated with a 33,000 dalton substance which can be dissociated into 29,000 and 4000-7000 dalton components.