Studies on Tetanus Toxin and Toxid. II. Isolation and Characterization of the Tetanus Toxin and Toxoid

Abstract

SummaryCrude tetanus toxin and toxoid were prepared by methanol precipitation. The toxin was purified by a combination of TEAE·cellulose and Sephadex G·200 chromatography at pH values less than 6· o. The toxoid was purified by DEAE· cellulose at approximately neutral pH values. The nature and amount of amino acids of the culture medium which had condensed with the tetanus toxoid proteins during detoxification with formaldehyde was determined.

Amino Acid Analysis and Molecular Weight Determination of Tetanus Toxin

“…In several different purification procedures employing gel filtration on Sephadex G-100 (6,9,11,28), tetanus toxin was excluded from the gel as judged by both Lf and MLD determinations. This would indicate a molecular weight greater than 100,000.…”

Studies on Tetanus Toxin and Toxoid IV. Interaction of Formaldehyde with Tetanus Toxin

“…A solution of pure tetanus toxin was prepared as previously described (Dawson and Mauritzen 1967). Briefly, the method involved a precipitation of the crude toxin with methanol at low temperature from the culture filtrate prepared at pH 5• O.…”

“…Where a partially detoxified toxin was fractionated on DEAE.cellulose, the differences in toxicity between the fractions were examined by challenging groups of 5 mice with a dose range of 120, 60, 30, and 15 I-'g of protein (approximately equivalent to 40, 20, 10, and 5 L f units) and keeping them under observation for 30 days. The flocculation values were determined by the technique of Dean and Webb (1926) as previously described (Dawson and Mauritzen 1967), and these were maintained during the HCHO treatments within the expected error of the test (± 10%).…”

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).…”