386 IMMUNO-CATALYSIS 



injections of 8 mgm. of the yeast dehydrogenase at 3-day intervals. The 

 crystalHne enzyme, stored as a suspension of crystals in alkaline am- 

 monium sulfate solution, was dissolved in water and injected im- 

 mediately. Sera were obtained two weeks after the last injections. 

 Stronger antisera were obtained after a second series of injections two 

 months later. Two rabbits received as many as four series of in- 

 jections. Chickens were similarly used for the production of specific 

 anti-enzyme serum. 



Antibody levels of the sera were measured by the precipitin reaction, 

 using the conventional ring test, and by the inhibition of activity of a 

 given amount of the yeast dehydrogenase. Enzyme activity was fol- 

 lowed by following the reduction of diphosphopyridine nucleotide 

 (DPN) spectrophotometrically at 340 m/t. The per cent inhibition of 

 15 ju.g. of crystalline enzyme was determined using 0.05 ml. of anti- 

 serum. There was a fair correlation between the precipitin titer and 

 the degree of inhibition by antisera. 



Rabbit antisera to the yeast dehydrogenase did not inhibit the activity 

 of hexokinase isolated from the same strain of baker's yeast. Rabbit 

 and chicken antisera to the yeast dehydrogenase did not exercise any 

 inhibitory effect on dehydrogenase of muscle. 



One-tenth ml. of enzyme solution containing 1 5 /tg. of protein was 

 added to a reaction mixture (in the Beckman cell) consisting of 2.5 ml. 

 of cysteine-pyrophosphate buffer and 0.2 ml. of DPN (final concentra- 

 tion=1.27XlO~^ moles per ml.) at 25°. The reaction was started 

 by the addition of 0.2 ml. of a fresh 1 : 1 mixture of 5.3 per cent sodium 

 arsenate and triose phosphate (final concentration^ 1.27X10"'^ 

 moles/ml.). Unless otherwise stated, antiserum was added to the 

 above reaction mixture. Bimolecular rate constants were calculated 



1 X 



from the equation: K=— —? — v 



t a^^a-x^ 



a=initial concentration of triose phosphate and DPN 

 xr=the amount of reduced DPN formed in time t (minutes) 



K was found to decrease with time, but K values based on one minute 

 readings were proportional to enzyme concentration. The per cent 

 inhibition of enzyme activity was calculated from the one minute 

 mixture. 



Antigen-antibody combination, measured by the extent of inhibition, 



