The Purification of Toxin from Ct,ostr.idiijm Botulinum Type A

Until recently it could be said that the status of our knowledge of bacterial exotoxins had not changed since Roux and Yersin in 1888 (1) first obt,ained the filtrable diphtheria toxin. Indeed, Topley and Wilson in 1936 (2) stated, “We have not yet. succeeded in isolating any bacterial esotoxin in a chemically pure state, although a considerable degree of ooncentration has been attained by various methods of fractional precipit.ation.” The same year Eaton (3) brought to light the first proof that a bacterial toxin (diphtheria) was a single protein-like substance by reporting the isolation of the toxin in a purified form. In 1937, Pappenheimer (4) isolated and characterized a toxic protein which appeared to be identical with diphtheria toxin. The properties of t,his protein w&e almost identical with those of the substance isolated by Eaton. With the protein nature of one bacterial toxin est,ablished, work on ot.her bacterial toxins (streptolysin (5), scarlet fever (6,7), tetanus (S), and Clostridium Ootulinum type Al) has confirmed the validity of t,he concept that bacterial exotoxins are proteins. When a toxin has been identified as a protein, proof of its purity presents many complex problems. Kekwick and McFarlane (9) present three criteria of a stable chemical compound: constancy of chemical composition, homogeneity of physical properties, and constancy of solubility. In work with a biologically active protein, the first crit,erion should of course include constancy of chemical composition and activity. A few of these criteria have been applied to bacterial toxins; e.g., diphtheria toxin (10) and scarlet fever toxin (7). Previous work by Sommer (11) on Clostridium botulinurn type A tosin has yielded a preparation containing 250,000,OOO mouse m.1.d.” per gm. This toxin was prepared in a peptic digest medium by precipitation at pH 3.5 to 4.0 and resolution in sodium acetate buffer. Since washed dried organisms were found to contain 100,000,000 mouse m.1.d. per gm., Sommer (11) assumed that the chemically pure toxin must have a potency many times greater.