Reconstitution of Bordetella bronchiseptica dermonecrotic toxin from two polypeptides

Dermonecrotic toxin (DNT) purified from sonic extracts of Bordetella bronchiseptica was mildly trypsinized. The trypsinized preparations were reversibly dissociated into two polypeptides, with M_rs of approx. 75 000 (fragment 1) and approx. 118 000 (fragment 2), by treatment with 100 mM dithiothreitol and 6 M urea. The two fragments were separated by gel filtration on a Sephadex G-200 column from the dissociated DNT preparations. Each of the purified fragments did not show dermonecrotizing activity for guinea pigs. The two fragments were antigenically distinct, but had partial identity with the native toxin. Whole B. bronchiseptica DNT molecule indistinguishable from the native toxin, in terms of M_r, electrophoretical behavior, antigenicity, and toxicity, was reconstituted by mixing freshly purified and dialyzed fragments 1 and 2 in a molar ration of 1:1 after removal of urea and dithiothreitol by dialysis. The anti-‘fragment 1’ antiserum completely neutralized dermonecrotizing activity of the purified DNT, whereas the anti-‘fragment 2’ antiserum did not.