High-molecular-weight hemolysin of Clostridium tetani

Clostridium tetani excretes hemolysins of two size classes, a high-molecular-weight hemolysin (HMH), which was eluted near void volume of a Sepharose 6B column, and conventional tetanolysin (molecular weight, approximately 50,000). The total hemolysin activity in the culture supernatant increased sharply with growth of bacteria and remained at a high level during autolysis. The content of HMH, however, decreased from 41% at 4 h of culture to 0.4% at the early stage of autolysis. The cell bodies also exhibited hemolytic activity, 70% of which could be solubilized and separated into HMH and the 50,000 Mr tetanolysin as extracellular hemolysins. The activity ratio of HMH to the total solubilized hemolysins was 0.45, on the average, at 6 h of culture but was 0.23 at the middle of logarithmic growth. Partially purified HMH from both sources appeared as broken pieces of cytoplasmic membranes under an electron microscope. The ratio of proteins to phospholipids in HMH was found to 3.26, a value similar to that in cell membrane. The total cell hemolytic activity decreased by 90 or 75% upon addition of chloramphenicol or anti-tetanolysin serum, respectively, into a 6-h-old culture of bacteria. It is suggested that HMH is a complex of tetanolysin with a membrane fragment and releases the conventional tetanolysin during bacterial culture.