Purification and Some Properties of Tetanolysin

Abstract

Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050‐fold to 500 HU/μg of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate‐gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000±3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pis, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly adsorbed onto erythrocytes than θ‐toxin of Cl. perfringens.

Mechanism of tetanolysin-induced membrane damage: studies with black lipid membranes

“…Our preparations contain two isozymes of equal specific activity with isoelectric points at pH 6.1 and 6.4. These properties are similar to those described by Mitsui et al (13). The tetanolysin had a specific activity of greater than 106 hemolytic units (HU)/mg of protein.…”

Purification and characterization of Vibrio metschnikovii cytolysin

“…2), although it had no thiol-activated properties. The amount of cholesterol needed to inhibit the V. metchnikovii cytolysin was about 100 times that generally used to inhibit thiol-activated cytolysin (9,27,34,36,43). Similarly, Gray and Kreger (15) have found that V. vulnificus cytolysin is inhibited by a large amount of cholesterol, but Miyoshi et al (28) have reported that V. vulnificus cytolysin is inhibited by cholesterol at a concentration similar to that which inhibits thiol-activated cytolysin.…”

Interaction of .theta.-Toxin (Perfringolysin O), a Cholesterol-Binding Cytolysin, with Liposomal Membranes: Change in the Aromatic Side Chains upon Binding and Insertion

“…Upon insertion into a membrane, soluble proteins, whose hydrophilic side chains are outside and hydrophobic side chains inside, are expected to change their conformations so that the hydrophobic side chains become exposed to the membrane lipid bilayer (van der Goot et al, 1992). Since &toxin and related thiolactivated cytolysins remain in a monomer form in solution Morgan et al, 1993) and are detected as oligomers in the membrane-inserting form as judged by electron microscopy (Sekiya et al, 1993;Mitsui et al, 1979), they are expected to undergo conformational changes during the membrane insertion process. Spectroscopic approaches, such as CD and fluorescence, are effective methods by which to analyze the structure of &toxin in connection with its cytolytic mechanism.…”