ANTI-ENZYME IMMUNITY 347 



(1941b) developed a refined technique to determine the concentration 

 of a-toxin in solutions by measurement of the turbidity developed. 



Manometric Method. A manometric method for the measurement of 

 lecithinase activity has been worked out by Zamecnik, Brewster and 

 Lipmann (1947). Lecithinase catalyzes the hydrolysis of lecithin into 

 phosphocholine and oleyl-stearyl-diglyceride. The hydrogen ion from 

 the former reacts with bicarbonate ion liberating carbon dioxide into 

 the gas phase. Electrometric titration of phosphocholine revealed a 

 pKo of 5.6. In a bicarbonate-carbon dioxide buffer system of pH 6.74, 

 approximately 95 per cent of the previously formed choline phosphoric 

 acid immediately reacted with bicarbonate ion, liberating carbon 

 dioxide into the gas phase. In a comparison between the acid-soluble 

 phosphorus method and the manometric method, a correction for the 

 latter method, amounting to not more than 10 per cent, was in- 

 dicated. 



f. Phenomenon of Opalescence in Serum and Lecitho-Vitellm 

 Produced by the Action of the Toxin of CI. Welchii. SeifFert (1939) 

 and Nagler (1939) reported that toxin of CI. welchii (CI. ■perfringens') 

 was capable of producing an obvious opalescence in some samples of 

 human serum both from normal and diseased persons. No effect was 

 observed with toxoid. A reinvestigation of this phenomenon by Mac- 

 Farlane, Oakley and Anderson (1941) resulted in the following find- 

 ings. They found that when CI. welchii toxin was mixed with some 

 human sera a very marked opalescence due to fat was observed within 

 a few hours. A clear filtrate of lecitho-vitellin prepared by heating a 

 saline suspension of egg-yolk and filtering through a Seitz-filter was 

 found to be extremely stable so long as it remained sterile. When this 

 crude lecitho-vitellin solution was treated with CI. welchii toxin, even 

 in the cold, opalescence appeared within a few minutes, proceeding 

 to flocculation and separation of a thick curd of fat. If lecitho-vitellin 

 is extracted with ether and the ether-extracted material is then treated 

 with the toxin, a much reduced opalescence occurred. The extraction 

 of the opalescent mixture with ether yielded a fat. 



The greater part of the opalescence produced in crude lecitho- 

 vitellin was due to the aggregation of finely divided free fat, a small 

 part of which was stated to be due to the setting free of fat from 

 compounds, probably lipo-proteins, as some proteins were denatured 

 at the same time. 



