The Purification and Concentration of Diphtheria Toxin

Abstract

Various methods of purifying diphtheria toxin are compared and the objections to the use of acid in any purification procedure are noted. A new method of purifying diphtheria toxin by precipitation with aluminum ammonium sulphate and cadmium chloride is described. By this method purified toxins containing 0.0005 mgm. N per Lf unit and 0.00002 mgm. N per M.L.D. have been obtained. Certain modifications of older methods such as acid precipitation and Al(OH)3 absorption gave lower yields of purified toxins containing also about 0.0005 to 0.0010 mg. N per Lf unit, but these preparations had a lower toxicity than those obtained by the new method. The ratio M.L.D./Lf is generally between 20 and 40 in crude toxins and toxins purified by various methods.[long dash]2. Diphtheria toxin may be successfully purified only by methods which involve protein precipitation or adsorption reactions. With progressive purification the ratio of N to Lf units in the toxin preparations appears to approach a constant value near 0.0005 mgm. N per Lf unit. None of the several purification procedures so far used has yielded toxins with a lower N/Lf ratio. The highly purified toxin consists mainly of a protein which contains no cysteine S, and very little tryptophane, but coagulates with heat and acid and gives definite biuret and other protein reactions. Traces of proteoses from the culture medium and of other proteins probably derived from the bacterial cells are also present. Toxin and bacterial protein are chemically and immunologically distinct. The chemical, immunological and physiological properties of diphtheria toxin observed during the course of this work indicate that it is a single protein-like substance.