136 THE BIOCHEMISTRY OF B VITAMINS 



The activation of a "zymase" system has been the classical method 

 employed in determining the concentration of diphosphopyridine nucleo- 

 tide, since the preparation of the apozymase can be readily accom- 

 plished. 34 The repeated extraction of dried brewers' yeast gives a residue 

 which contains all the components of the alcohol fermentation system 

 except diphosphopyridine nucleotide. In the presence of the apozymase 

 preparation the rate of fermentation (measured by following the evolution 

 of carbon dioxide) gives a direct measure of the amount of coenzyme 

 introduced into the system. In this process the coenzyme is needed to 

 accept hydrogen atoms from glyceraldehyde-phosphate and subsequently 

 to donate them to acetaldehyde. Aerobic processes (such as oxidation of 

 lactic acid 35 and malic acid) 27 have also been used for the enzymatic 

 estimation of diphosphopyridine nucleotide. In addition to the appro- 

 priate apoenzyme and substrate the system must contain the other 

 enzymes needed for accepting the hydrogen atoms from the reduced 

 coenzyme and transporting them to the final hydrogen acceptor. If molec- 

 ular oxygen is the acceptor, the reaction can be followed manometrically ; 

 if methylene blue is used, the rate of decolorization is measured. 



The oxidation of glucose-6-phosphate (Robinson ester) to 6-phospho- 

 gluconic acid is catalyzed by a dehydrogenase (Zwischenferment), the 

 coenzyme of which is triphosphopyridine nucleotide. 36 This reaction can 

 be used as a specific method for the determination of this coenzyme, 

 since the diphospho nucleotide cannot act here as the hydrogen acceptor. 

 The reaction can be followed in several ways depending upon the hydro- 

 gen transporting systems to which it is coupled. The most sensitive and 

 accurate method is one in which cytochrome-c is the component finally 

 reduced. 37 The rate of reduction of cytochrome-c can be easily followed 

 spectrometrically. By this procedure quantities as small as 0.02 micro- 

 gram of the coenzyme can be measured. 



Hemophilus influenzae and Hemophilus para-influenzae cannot syn- 

 thesize the nicotinamide nucleoside from nicotinic acid or nicotinamide; 38 

 consequently, these organisms do not respond to nicotinic acid or nico- 

 tinamide but must have an exogenous supply of either of the coenzymes 

 or certain degradation products in which the pyridine-ribose bond is 

 intact. 39 These organisms have been used to assay for the coenzymes by 

 conventional microbiological procedures. Although this method lacks 

 specificity because some degradation products are active, the interference 

 by such substances in most instances probably is negligible. 



The chemical reagents employed in the chemical determination of the 

 free vitamin also react with the coenzymes, and cannot be directly used 

 to distinguish between the simple vitamin and its more complex deriva- 

 tives. However, chemical methods can be of use when it is known that 



