ANTIBODIES AGAINST RESPIRATORY ENZYMES 387 



was complete within the 10 minute period allowed. The turbidity 

 developing in the Beckman cell due to antibody-enzyme flocculation 

 also reached a maximum during this period. The absorption at 340 m/i. 

 due to the presence of a precipitate was small and was corrected for 

 the initial reading. 



It was found that with increasing amounts of antiserum the degree 

 of inhibition increased almost linearly up to about 80 per cent. Beyond 

 this the inhibition leveled off, never reaching complete inhibition. 

 The residual enzyme activity in the presence of an excess amount of 

 antibody suggested that the insoluble enzyme-antibody complex itself 

 retained activity amounting to about 10 per cent of the original enzyme 

 activity. The removal of the precipitate by centrifugation left a 

 supernatant fluid with no enzyme activity, while the washed precipitate 

 showed an activity only slightly lower than the residual activity. 



Effect of Di'phos'pho'pyndine nucleotide (DPN^ on Enzyme-Anti- 

 hody Combination. As discussed above the presence of an excess 

 amount of antiserum in the reaction system caused up to 95 per cent 

 inhibition of the enzyme activity. The residual 5-10 per cent activity 

 was that of the enzyme-antibody precipitate, which showed no evidence 

 of dissociation at increasing concentration of DPN and triosephosphate. 



In systems where only 0.05 ml. of antiserum was added to the re- 

 action mixture containing DPN, enzyme, buffer etc., the activity de- 

 termined after 5 minutes incubation showed 31 per cent inhibition of 

 the enzyme. Longer incubation did not increase the extent of inhibi- 

 tion. When DPN was absent during the preliminary incubation of 

 antiserum and enzyme, there was found 54 per cent inhibition of 

 activity. The substrate triosephosphate had no influence on this rela- 

 tionship among the three reactants, coenzyme DPN, enzyme and 

 antibody. 



The rate of combination of enzyme with antibody was determined 

 while the enzyme was acting on its substrate. High concentrations of 

 triosephosphate and DPN were used, and under these conditions the 

 rate was found to be nearly linear after the first half minute. Rabbit 

 antiserum in deficient and excess amounts was added to identical 

 reaction mixtures at the third minute and the rate followed until it 

 again became linear, indicating completion of enzyme-antibody com- 

 bination. Under these conditions, the difference between the control 

 rate and the residual rate of the system containing an adequate amount 



