Methods for the purification and crystallization of tetanal toxin are described. The methods consist of the multiphase fractionation system involving methanol as the organic precipitating agent under controlled conditions of pH, ionic strength, protein concentration, and temperature.

Crystalline tetanal toxin has an electrophoretic mobility of 2.8 x 10–5 in veronal buffer of 0.1 ionic strength at pH 8.6. The solubility of freshly prepared toxin is essentially constant. The isoelectric point is 5.1 ± 0.1. The crystalline toxin contains 1 per cent sulfur, traces of phosphorus, and gives the usual protein reactions. It does not contain carbohydrate.

The crystalline toxin does not precipitate anti-Clostridium tetani rabbit serum. The final product contains between 3400 and 3600 Lf and about 6.6 x 107 M.L.D. per mg. N.

Crystalline tetanal toxin is spontaneously converted to a flocculating atoxic dimer upon standing at 0°. This change is accompanied by the appearance of another molecular species as judged by constant solubility tests. Ultracentrifugal analysis of these fractions reveals that tetanal toxin has a sedimentation constant of 4.5 Svedberg units while the atoxic flocculating dimer sediments at 7 S.

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