PURIFICATION AND CHARACTERIZATION OF INTRACELLULAR TOXIN-A OF CLOSTRIDIUM-DIFFICILE

After sonic disintegration of Clostridium difficile cells, intracellular toxin A was purified to homogeneity by thyroglobulin affinity chromatography (TGAC) followed by anion-exchange (Mono Q) by fast protein liquid chromatography (FPLC). High haemagglutinating (HA) activity was detected in TGAC-unbound fractions (2(9)/50 mul), but not in TGAC thermal eluates (2(0)150 mul). The low HA titre of the thermal eluates was markedly increased to 2(5)150 mul after dialysis against 0.02 m Tris-HCI (pH 7.5). A disparity in the position of the peaks containing cytotoxic and HA activity was observed in the first Mono Q-FPLC step. Intracellular toxin A without HA activity was obtained by a second Mono Q-FPLC step. The M(r) of the intracellular toxin A was estimated by polyacrylamide gel electrophoresis (PAGE) to be 580 kDa under non-denaturing conditions. The minimum doses of the toxin causing cytotoxicity, mouse lethality and enterotoxicity were 0.83 ng, 8.7 ng and 5 mug, respectively.