Tetanus Toxin and Antigenic Derivatives I. Purification of the Biologically Active Monomer

Abstract

A procedure for the isolation of pure tetanus toxin in a lethal monomeric form was developed based on the extraction of whole cells and chromatographic techniques. A crude extract of toxin was obtained by hypertonic extraction of cells from a 72-hr culture of Clostridium tetani Massachusetts strain. The extract was precipitated with ammonium sulfate and further purified by sequential use of ion-exchange chromatography and gel filtration. The degree of purification obtained by the fractionation procedures was monitored by polyacrylamide gel electrophoresis. The pure toxin has an average specific activity of 150 × 10 6 mouse MLD per mg of N and 3,000 Lf per mg of N. Immunological purity was demonstrated by a single line on both immunoelectrophoresis and agar double diffusion. One band was obtained on polyacrylamide electrophoresis, as was a single symmetrical peak in the ultracentrifuge and on Sephadex G-100 chromatography. The pure protein has an absorbancy ratio (280/260 mμ) of 2.1 in phosphate buffer ( p H 7.5).

Tetanus Toxin and Antigenic Derivatives II. Effect of Protein and Formaldehyde Concentration on Toxoid Formation

“…Analytical procedures. Polyacrylamide gel electrophoresis was done, and flocculation titers (Lf) were obtained as previously described (5). Agar double diffusion was performed according to Ouchterlony (6).…”

Amino Acid Analysis and Molecular Weight Determination of Tetanus Toxin

“…This would indicate a molecular weight greater than 100,000. Other investigators (14,19,27) have found the majority of the flocculating material to be eluted immediately after the excluded fraction. Salenstedt and Tirunarayanan (28), using partially purified toxin that had been conconcentrated only by ultrafiltration, found that the toxin, as determined by toxicity tests in mice, was eluted in the excluded fraction of Sephadex G-200.…”

“…Murphy and Miller (19) noted that upon standing their 7S toxin lost toxicity with no observable change in molecular weight. This would suggest that the loss in toxicity was due to a small molecular change rather than polymerization such as that suggested by Pillemer et al (24).…”

“…However, the preparations obtained have not been shown to be a single component by the criteria of immunodiffusion, immunoelectrophoresis, and analytical disc gel electrophoresis. Murphy and Miller (19) reported purifying tetanus toxin meeting these specifications by using a combination of gel filtration and diethylaminoethyl (DEAE) cellulose chromatography, but the toxin so obtained was not characterized further. Utilizing both precipitation techniques and column chromatography, Bizzini et al (2) purified, partially characterized, and compared tetanus toxin obtained by extraction of the bacterial cells with that obtained after excretion into the culture medium.…”

Tetanus toxin.

“…In the last two decades, the toxin has been isolated in a homogeneous state (12,21,22,52,65,73), and its physical and chemical characteristics have been defined in various laboratories in different parts of the world (5,12,14,20,22,50,52,63,64,73,83,85; S. G. Murphy, T. H. Plummer, and K. D. Miller, Fed. Proc., p. 268, 1968).…”