Properties of the Cholera Exo-Enterotoxin: Effects of Dispersing Agents and Reducing Agents in Gel Filtration and Electrophoresis

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RESUMO

Initial studies, by using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), led to the attractive hypothesis that the cholera exo-enterotoxin (choleragen) consisted of two noncovalently linked peptides of 56,000 and 28,000 molecular weight. The spontaneous toxoid (choleragenoid) appeared to be identical with the 56,000 molecular weight piece. The results appeared to be very similar to those obtained recently with diphtheria toxin. However, they did not agree with earlier studies which indicated that both the toxin and the toxoid consisted of subunits of approximately 14,000 molecular weight. More extensive investigation, by using gel filtration with 3H-labeled toxin, ultracentrifugation, and immunologic studies, failed to support the observations of SDS-gel electrophoresis, suggesting the existence of a 28,000 molecular weight fragment which is unique to the toxin. The results were more compatible with our earlier observations regarding subunit composition. It is concluded that these proteins are relatively resistant to dissociation by SDS and need to be unfolded by prior treatment for more complete dissociation into subunits. Indirect evidence suggests that the 28,000 fragment may be a dimer, or larger aggregate, of a smaller subunit which can be integrated in the formation of choleragenoid. It is also evident that caution should be exercised in the interpretation of results, however pleasing, obtained only by SDS-gel electrophoresis.

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