120 A SYMPOSIUM ON RESPIRATORY ENZYMES 



based on the coenzyme properties of adenylic acid and its homo- 

 logues (cophosphorylase) in the enzymatic spHtting of phospho- 

 pyruvic acid (see formula 11). In the absence of a suitable acceptor 

 the phosphoric acid appears in a free state in an amount propor- 

 tional to the amount of cophosphorylase present in the system; by 

 this method 10 to 100 micrograms of adenylic acid can be deter- 

 mined (35). 



Methods of Determination 



Only the methods based on the coenzyme properties will be dis- 

 cussed here. For determining the presence of cozymase Harden (2) 

 originally used a press extract of yeast as a source of apoenzymes 

 and a boiled yeast extract as a source of coenzyme. A great im- 

 provement over this method was made when Euler and Myrback 

 found that dried brewer's yeast loses its cozymase by repeated ex- 

 traction with cold water (le, 36). The remaining product was found 

 to contain all the apoenzymes, activators, and coenzymes necessary 

 for fermentation except cozymase. Therefore it was called apo- 

 zymase. The use of this preparation is still the simplest and perhaps 

 the most accurate method of determination, its accuracy being Hm- 

 ited only by the errors of the manometric measurements. Another 

 test system recently elaborated by Jandorf , Klemperer, and Hastings 

 uses, instead of the function of cozymase in fermentation, the co- 

 enzyme properties of cozymase in glycolysis, according to the 

 following scheme (37): 



Hexose diphosphate -^ phosphoglyceraldehyde + dihydroxy- 

 acetone phosphate 



Phosphoglyceraldehyde + H3ASO4 -^ arsenophosphoglyceral- 

 dehyde 



Arsenophosphoglyceraldehyde + DPN -> arsenophospho- 

 glyceric acid + DPN-Hg 



Arsenophosphoglyceric acid -^ 3-phosphoglyceric acid + 

 H3ASO, 



Phosphoglyceraldehyde + DPN-Ho -> glycerophosphoric 

 acid + DPN 

 6) Hexose diphosphate -^ 3-phosphoglyceric acid + glycero- 

 phosphoric acid 



The amount of phosphoglyceric acid produced in a given time in 

 the presence of bicarbonate buffer can be measured manometrically 

 with the Warburg apparatus. The test system for codehydrogenase 

 II as given by Warburg uses the dehydrogenation of Robison ester 



