Haemolytic activity of Escherichia coli

Production of large amounts of high titre E. coli haemolysin has been achieved in a chemically-defined medium and in a glucose-nutrient broth medium. In chemically-defined medium, only cell-associated, (3-haemolysin was produced; in nutrient broth both extracellular a-haemolysin and p-haemolysin were found and, maximum yields of a-haemolysin were obtained within 2 hr after inoculation of cultures. More a-haemolysin was produced when large initial inocula were used but, loss of haemolytic activity occurred rapidly after maximum levels were reached. Evidence is presented which suggests that a-haemolysin is a "released" form of p-haemolysin. Large molecular weight proteins, contained in nutrient broth, enhanced levels of a-haemolysin without affecting growth; both forms of haemolysin required calcium ions for activity, were inhibited by incubation with trypsin and were not affected by thiomersalate (in the case of p-haemolysin inhibition of haemolysis by thiomersalate was not observed after haemolytic E, coli cells had adsorbed to sheep erythrocytes). Highly purified a-haeraolysin was obtained by precipitation methods using 50% (w/v) ammoniun sulphate (stage l), and dialysis against 0.005M acetate buffer, pH 4.6 (stage II), and by gel filtration on Sephadex G-200 at pH 7.5 an eluant buffer containing 0.01M Tris, 0.1M NaCl and 5% (w/v) glycerol (stage III). No loss of haemolytic activity was observed following dialysis at any stage in the purification. Other purification procedures, such as electrofocusing, proved of little practical value. Using the above procedures, J6% of the total activity contained in crude culture filtrates was recovered and a 4000-fold increase in specific activity was achieved. This degree of purification has not previously been reported for E. coli haemolysin. D-urine precipitation procedures, activation of haemolytic activity occurred. Furthermore, in eluant buffer containing: glycerol, the haemolysin was eluted from Sephadex G-200 near the void volume as two closely associated, but distinct peaks of activity. Several techniques were employed to estimate the molecular weight of a-haemolysin. Diffusion coefficient analysis (D20 = 2.4 x 10