122 A SYMPOSIUM ON RESPIRATORY ENZYMES 



H. para- influenzae have been found to require cozymase or code- 

 hydrogenase II. According to LwoflF only 0.004 microgram per milli- 

 liter of peptone solution is necessary to produce visible growth under 

 standard conditions. The method does not distinguish between co- 

 dehydrogenases I and II. Dihydrocozymase was found to be in- 

 ferior as a nutrilite to an equivalent amount of cozymase (42). This 

 may be due to a difference in the permeability of the cells to the two 

 compounds. 



For the examination of pure or almost pure coenzyme prepara- 

 tions the spectrophotometric determination of the dihydro com- 

 pounds is a very accurate method. The solutions must, however, be 

 free from impurities that absorb in the ultraviolet region in which 

 the dihydrocoenzymes exhibit their characteristic absorption. The 

 chemical methods of determination cannot be discussed here in 

 detail. 



The Apodehydrogenases 



Since the history of the apodehydrogenases has been reviewed in 

 several comprehensive articles (la, c, g, 43a-d), they will simply be 

 listed here, and our present knowledge about their purification, na- 

 ture, and function will be summarized and their coenzyme speci- 

 ficity discussed. 



As can be seen from Table 7, some of the apodehydrogenases have 

 been prepared in a pure state and obtained in a crystalline form. 

 The main progress in the field was made by Warburg and his co- 

 workers, especially Christian, Negelein, Gerischer, Haas, Wulff, 

 and Kubowitz. Warburg's methods for purification consist mainly 

 in precipitations of the apoenzymes at the isoelectric point, their 

 fractional precipitation by ammonium sulfate or organic solvents, 

 and removal and inactivation of other enzymes by heat denatura- 

 tion at a temperature of about 50° C. Of special interest is the pre- 

 cipitation of the diphosphoglyceraldehyde apodehydrogenase in a 

 step of its purification by addition of nucleic acid (44), 



The isolation of the apodehydrogenases in a pure state was im- 

 portant for a detailed study of their relation to coenzymes and sub- 

 strate by the spectrophotometric technique. Whereas the use of 

 crude apoenzymes does not exclude the possibility of more com- 

 plex reactions, a definite conclusion can be drawn respecting the 

 mechanism of a reaction if pure apoenzyme, coenzyme, and sub- 

 strate are used. Our previous conception of the function of cozymase 

 in the dehydrogenation of triosephosphate was greatly revised in 



