254 IMMUNO-CATALYSIS 



1 mg. papain injected intravenously. Normal guinea pigs showed no 

 anaphylactic symptoms on receiving 5 to 10 mg. papain preparation. 

 Using the Schultz-Dale technique the uterus of sensitized guinea pigs 

 contracted strongly in the presence of 1 mg. papain. In precipitation 

 tests antipapain rabbit sera reacted with papain but not with pepsin. 

 Antipepsin sera reacted weakly with pepsin but showed no reaction 

 with papain. In complement fixation experiments antipapain serum, 

 in 1 : 20,000 dilution, inhibited the hemolysis of red cells. 



In view of the fact that papain used by Haas was perhaps not a 

 single entity, or may have been contaminated with other protein im- 

 purities, the above results may not be taken as conclusive evidence 

 that papain itself was responsible for these reactions. The following 

 experiments which deal with the inhibition of the proteolytic activity 

 of the papain preparations with specific immune rabbit sera show con- 

 clusively, however, that a specific antibody against papain was pro- 

 duced in the blood of rabbits immunized with these preparations. 



Inhibition of the Proteolytic Activity of Papain with Antipapain 

 Sera. The maximum antigen-antibody reaction indicated by the 

 amount of precipitate produced was found to cause the highest degree 

 of inhibition of the proteolytic activity. 



Four ml. of inactivated serum (immune or normal serum) treated 

 with I ml. of 2 per cent papain solution were incubated at 40° for one 

 hour. The mixture, after treating with 1 ml. of 0.2 M disodium citrate 

 solution and 0.4 ml. of 0.5 per cent potassium cyanide solution (neu- 

 tralized with 0.16 N HCl solution against methyl red) was made up 

 to a volume of 20 ml. with distilled water. To this was added 4 ml. of 

 5 per cent casein or 4 ml. of 3 per cent gelatin solution of pH 4.8 to 

 4.9. The reaction mixture was incubated at 37°C. for 44 hours. At 

 the end of 20 and 44 hour periods sterile samples were taken and 

 titrated with alcoholic 0.01 N sodium hydroxide solution to determine 

 the increase in acidity as a result of proteolysis. 



In this series of experiments papain-antipapain precipitate was left 

 in the reaction mixture. In another series of experiments the precipi- 

 tates resulting from the papain-antipapain reaction were centrifuged 

 and 5 ml. of the clear supernatant solution was tested for proteolytic 

 activity as described above. In all these experiments reaction mixtures 

 were held under sterile conditions. The results showed that, in contrast 

 to the results obtained in the presence of normal rabbit sera, the 



