Production, purification, and composition of staphylococcal alpha toxin.

Coulter, John R. (Institute of Medical and Veterinary Science, Adelaide, Australia). Production, purification, and composition of staphyloccocal alpha toxin. J. Bacteriol. 92:1655-1662. 1966-Pure staphylococcal alpha toxin has been prepared in quantities suitable for chemical, biological, and clinical characterization. Purification was achieved by acid-methanol precipitation, chromatography on G100 Sephadex, and electrophoresis in G100 Sephadex. We recovered 25% of the crude toxin in pure form, a yield of 12 mg/liter of crude culture supernatant fluid. The pure material gave a single line on gel diffusion and on immunoelectrophoresis and gave a single symmetrical peak in the ultracentrifuge. The alpha toxin was highly unstable, with a half-life of 3 days at 0 C (pH 7.8); solutions of it could not be frozen, and we found no method to stabilize it. On standing, a thready precipitate appeared; it was inactive against rabbit red cells, was not lethal to rabbits, but was able to elicit specific anti-alpha antibody production in the rabbit. There is evidence that alpha toxin is an associating molecule, with a mean sedimentation coefficient of approximately 3.0 and a molecular weight of approximately 30,000. The lowest molecular weight, found by equilibrium ultracentrifugation, was 21,200 +/- 400. The amino acid composition was determined, and the high positive charge was explained by the presence of lysine, arginine, and histidine, and by amination of the aspartic and glutamic acid residues. Histidine and arginine were shown to be N-terminal amino acids, a fact which suggests the presence of two polypeptide chains. No carbohydrate was present. The ultraviolet absorption spectrum showed a maximum at 274.5 mmu, a minimum at 251.5 mmu, and a shoulder at 292 mmu. The toxin was without proteolytic or phospholipase activity, and its highly specific action on cell membranes still remains unexplained.