Purification and characterization of Aeromonas hydrophila beta-hemolysin

Aeromonas hydrophila hemolysin was excreted in our culture conditions during the stationary growth phase. The toxin was purified to homogeneity by a three-step method: ultrafiltration, acid precipitation in the presence of RNA and anion exchange chromatography with FPLC apparatus. Beta-hemolysin is a protein not associated with lipids, carbohydrates or nucleic acids whose subunit mol. wt is 51,000. The mol. wt determined by polyacrylamide gel electrophoresis suggests that the molecule is in a trimeric form. The toxin is thermolabile and inactivated by proteolytic enzymes such as trypsin, chymotrypsin, pronase, subtilisin and proteinase K. Antibodies raised against the beta-hemolysin neutralize both hemolytic and cytotoxic activities. When injected at high dose, this purified hemolytic protein causes a positive rabbit ileal loop test, thus indicating that beta-hemolysin could be the main virulence factor involved in intestinal symptoms.