ANTI-ENZYME IMMUNITY 349 



support as discussed below from the findings of Maschmann (1937) 

 that the toxin of CI. welchii (_Cl. -perfringens^ also exercises a strong 

 proteolytic activity (p. 256). 



g. Production of Opalescence in Serum and Lecitho-Vitellin as a 

 Measure of the Potency of the Toxin. As discussed previously, Seiffert 

 and Nagler observed that the toxin of CI. welchii produces an obvious 

 opalescence in some samples of human serum. This effect was also 

 produced by the action of toxin on lecitho-vitellin. It is evident that 

 enzymatic action of the toxin produces definite chemical changes in 

 the constitution of the above mentioned lipo-proteins. This phenome- 

 non has been studied in some detail, as discussed above, by 

 MacFarlane, et al. They observed that any process which reduced the 

 hemolytic activity of the toxin also reduced its capacity to produce 

 opalescence in serum and lecitho-vitellin. This strict parallelism be- 

 tween hemolysis by a-toxin and the production of free lipoid from 

 lecitho-vitellin— a known lipo-protein— led these authors to suppose that 

 these activities might be manifestations of the same enzyme reaction, 

 since hemolysis may well follow the breakdown of the lipo-protein 

 complex in the red cell envelope. 



As the hemolytic activity of the toxin was neutralized by the spe- 

 cific antitoxin, so was the enzymatic activity of the toxin, responsible 

 for the production of opalescence in lipo-proteins, neutralized by the 

 same antitoxin. The potency of the antitoxin in producing these two 

 effects ran parallel. Neither the hemolytic activity nor the production 

 of opalescence were neutralized by antitoxins prepared against other 

 toxins. These facts showed a high degree of identical specific action 

 characteristic for both the enzyme and antitoxin reactions. 



The hemolysis and the production of opalescence by a-toxin were 

 found to be dependent on the presence of calcium ions. In the absence 

 of calcium, both reactions were absent. Potassium oxalate, disodium 

 phosphate and sodium citrate, which form non-ionizable salts with 

 calcium, prevented both hemolysis and opalescence, suggesting that 

 ionized calcium, which was found by MacFarlane and Knight (1941) 

 to be an accelerating factor of lecithinase activity, might play an es- 

 sential part in the reaction. The restoration of calcium to the system 

 led to the restitution of all of the in vitro activities of the toxin. 



Another point which requires emphasis here is the relation of the 

 proteolytic activity of the toxin to the phenomenon of opalescence. 



