ANTI-ENZYME IMMUNITY 345 



calcium ion, essential for the lecithinase reaction, from the field of 

 reaction due to the formation of Ca-phosphatidyl serine. The structural 

 relationships of lecithin to the above substances are shown on page 344. 



d. Characteristics of Lecithinase. Lecithinase activity as described 

 above was estimated (MacFarlane and Knight, 1941) by the rate of 

 formation of acid-soluble phosphorus from an aqueous emulsion of 

 lecithin, by the glycerinated toxin. Under the conditions of the ex- 

 periment no hydrolysis of lecithin took place in the absence of toxin at 

 pH 7.1 and 37°C. or in trichloracetic acid at room temperature for 

 30 minutes. None of the toxins examined hydrolyzed diphenyl-, 

 monophenyl-, glycero-phosphate or nucleic acid; the enzyme is there- 

 fore a lecithinase. 



The dilute enzyme is very rapidly inactivated by the exposure of a 

 shallow layer, by shaking or by bubbling air through it. This inac- 

 tivation was assumed to be due to surface denaturation and not to 

 oxidation, for the enzyme was unaffected by treatment with 0.3 per 

 cent hydrogen peroxide solution, while bubbling with purified nitro- 

 gen (passing through alkaline Na2S204) inactivated it. The percentage 

 of inactivation of toxin was determined by animal test and by its action 

 on egg-yolk. 



Toxin lecithinase was found to be comparatively heat-stable, 45 

 per cent of the activity remaining after heating in borate buffer of pH 

 7.6 for 10 minutes at 100° in a sealed ampoule. It was more rapidly 

 inactivated in acid than in alkaline solution at this temperature. The 

 enzyme was active over a wide pH range of 7.0 to 7.6, in borate buffer; 

 the activities at pH 9.3 and 5.2 in borate and acetate buffers were, 

 respectively, 64 and 66 per cent of the activity at optimal pH. The 

 activity of the enzyme was greatly affected by the presence of calcium 

 ions. 



Low concentrations of magnesium salts also activated, but higher 

 concentrations of 0.01 M or more inhibited the enzyme. 



Toxoids had no lecithinase activity, nor did they influence the 

 lecithinase activity of toxins. The lecithinase activity of traces of 

 a-toxin present in dry type C toxin was not inhibited by the presence 

 of ^- and 8-toxins in type C toxin. 



e. Quantitative Methods Used for the Measurement of Lecithin- 

 ase Activity: Acid-Soluble Phosphorus Method. MacFarlane and 

 Knight (1941) measured the rate of formation of acid-soluble 



