Purification and Some Properties of Clostridium Welchii Type A Theta Toxin

Abstract

Theta toxin has been highly purified by fractionation of filtrates of Clostridium welchii type A in methanol-water mixtures under controlled conditions of pH, ionic strength, protein concentration, and temperature. By means of six fractionation steps, a product containing 2,450,000 theta hemolytic units per mg of protein nitrogen was obtained. This preparation was free of collagenase and hyaluronidase and contained less than 0.01% of the total amount of lecithinase activity of the parent filtrate. As judged from electrophoretic analysis, a value of 2,650,000 theta hemolytic units per mg of protein nitrogen was calculated for pure theta toxin. The degree of purification depended upon the potency of the parent filtrate and upon the relative ratio of theta toxin hemolytic activity to lecithinase Lb activity which it contained. Although no substrate other than the red blood cell has yet been found for theta toxin, certain of the properties of the purified hemolysin indicate that it might act enzymatically. The exceedingly small amount of activated hemolysin which produces lysis suggests its action is catalytic. About 2.5 × 10^-6 mg of protein represents one hemolytic unit. This amount of hemolytically active protein lysed 6 ml of a 0.25% suspension of sheep red blood cells in 30 minutes at 37°C. It is improbable that such a small amount of protein could alter this concentration of red cells other than by catalytic action. The protein nature of theta hemolysin, its activation by —SH compounds, its pH and temperature dependence, also suggest that theta toxin is an enzyme. The manner in which activated theta toxin brings about hemolysis of red cells could be explained by the enzymatic attack of some constituent of the red cell membrane. However, the identity of this constituent is not known. Positive proof that theta toxin is an enzyme must await the identification of its specific substrate.