The Purification and Concentration of Diphtheria Toxin

Abstract

The purpose was to determine what proportion of purified diphtheria toxin consists of bacterial protein. Bacterial precipitinogens were detected and measured by precipitin tests with antibacterial serum prepared by immunizing rabbits with washed diphtheria bacilli. Measurements by the precipitin test of the amt. of bacterial protein in purified toxin were based on titrations of the antibacterial serum against preparations containing known amounts of bacterial protein. The bacterial protein constitutes 0.5-2% of the total protein in the most highly purified preparations. Slightly denatured toxin is precipitated at a higher pH, and a lower conc. of (NH4)2SO4, and is more readily adsorbed on magnesium or aluminum hydroxide than is undenatured toxin. Evidence is presented that the porphy-rins found in crude and purified toxins are not combined with protein but are precipitated near the iso-electric point of toxin and are easily adsorbed on protein precipitates.[long dash]The results indicate that the purified protein, which has been freed of all but small traces of impurities detectable by the methods employed, is diphtheria toxin.