Immunochemical Studies of Two Cholera Toxin-Containing Standard Culture Filtrate Preparations of Vibrio Cholerae

Two crude toxin preparations of Vibrio cholerae, labeled lot 4493 G (Inaba) and lot 001 (Ogawa), consisting of freeze-dried culture filtrate, were studied with regard to toxicity, precipitinogenicity, and chemical composition. Lot 4493 G contained much more carbohydrate and less protein than lot 001, but both of the preparations contained three precipitinogens which were identical. One of these factors was identified as cholera exotoxin. In addition, both contained a fourth cross-reacting precipitinogen related to lipopolysaccharide. The toxicity of the lots was very similar. The toxin active as permeability factor in the intradermal test, identified completely in comparative double-diffusion analyses with the toxin causing cholera-like symptoms in experimental animals, strongly indicated that the same toxin molecule was active in the two different model systems. The isoelectric point of the toxin was about pH 7, but some toxic activity focused toward a pH of 9. The toxicity was completely retained by a UM-10 membrane (cut at a molecular weight of 10,000), practically retained by a PSED membrane (cut at a molecular weight of 25,000), but not retained by an XM-50 membrane (cut at a molecular weight of 50,000). By gel filtration through agarose, it was possible to separate and purify more than 1,000-fold the toxin from the other, more rapidly filtering three precipitinogens. With lot 4493 G, the toxicity of the agarose gel filtration fractions was restricted to an elution volume similar to that of globular proteins with a molecular weight of 25,000 to 38,000, whereas gel filtration experiments through Sephadex G-75 indicated a size of the toxin corresponding to a molecular weight of about 55,000 to 60,000.