ANTI-ENZYME IMMUNITY 341 



dilutions. They stated that the zeta toxin of Prigge is identical with 

 the a-toxin of Glenny, et al. 



Van Heyningen (1941a) confirmed the above objection of MacFar- 

 lane, et al. by demonstrating that the hemolytic activity of a-toxin 

 (zeta of Prigge) requires the presence of calcium ion and that the 

 presence of phosphate buffer suppresses hemolysis by a-toxin. Phos- 

 phate buffer had no suppressing effect on hemolysis by ^-toxin. Van 



"'Comment on the So-Called Conversion of e-Prototoxin into e-Toxin of Ch welchii 

 Type D. Bosworth and Glover (1934-1935) observed that the culture filtrates or 

 solutions of dried "toxin" of Ch welchii D acted upon by small quantities of trjrpsin 

 became, after a short period, much more toxic. This phenomenon was prevented if 

 the trypsin was previously treated with normal "anti-tryptic" serum, apparently due to 

 the presence of trypsin inhibitor in the normal serum (see p. 163). Crude trjrpsin, 

 purified trypsin, and a "purified form of chymotrypsin" increased the activity of crude 

 type-D toxin, but pepsin, papain, diastase and amylase did not. Despite the increased 

 toxicity the combining power was not changed, and they considered this as additional 

 evidence that the results cannot be explained by the supposed presence of an inactive 

 proto toxin which is converted into the e- toxin by trypsin. 



Turner and Rodwell (1943a, 1943b) report that the cultures of most strains of 

 CI. welchii type D contain, in addition to e-toxin, an almost atoxic relatively ther- 

 mostable e-prototoxin with the same combining power as e-toxin, and that activation 

 is the result of proteolytic conversion of prototoxin into toxin. They suggest that in 

 type-D cultures e-toxin develops from prototoxin through the proteolytic action of 

 intrinsic protease. Their conclusions are listed here: (a) a precursor of e-toxin is 

 excreted from the cells during the growth phase; (b) this precursor is atoxic or 

 relatively so; (c) it is more thermostable than e-toxin; (d) it has the same capacity 

 to combine with specific globulin as toxin has; (e) it is equally antigenic; (f) the 

 groups to which it owes its toxicity are "masked" by something that can be digested 

 away by a variety of proteases, including type-D protease; and (g) e-toxin is derived 

 from e-prototoxin and is not excreted directly by the cells. 



It is to be noted that the e-prototoxin is antigenically complete but is atoxic. Its 

 power to combine with the antitoxin is the same as that of fully active toxin. During 

 the growth of the organism maximal combining power and potential toxicity are 

 reached in cultures after only a few hours' growth, whereas primary toxicity may not 

 become maximal for four or more days. The above observations show that the com- 

 plete toxin molecule is formed in its antigenically specific and potentially toxic form, 

 and, most likely, secreted from the cells in combination with another non-toxic protein 

 in the form of an atoxic molecular complex. It does not appear reasonable to assume 

 that such a complex conforms with the precursor idea. Since the maximal combin- 

 ing power and the potential toxicity are reached in cultures after only a few hours' 

 growth, it is reasonable to consider that these properties are inherent in the free 

 toxin molecule or the atoxic molecular complex. The proteolysis of the atoxic complex 

 eliminates the non-toxic protein from the atoxic complex, exposing the toxic groups 

 already present in the original toxin molecule. In another section of this monograph, 

 several examples are given showing clearly how indifferent proteins combining with 

 active viruses and enzymes form inactive and readily dissociable molecular com- 

 plexes. It would seem that Turner and Rodwell's e-prototoxin possesses features 

 similar to such protein-protein complexes. These considerations do not lend support 

 to the supposition that the atoxic complex is the primary product in the chemical 

 genesis of the fully developed toxin molecule. 



