COENZYMES DERIVED FROM B VITAMINS 135 



compound would yield an esterified derivative of adenylic acid which 

 would be inactive when tested for "cophosphorylase" activity. 24 



An enzyme occurring in rabbit tissue has been shown to cleave the pyro- 

 phosphate linkage in diphosphopyridine nucleotide to produce the two 

 mononucleotides, 248 and an enzyme preparation has been obtained from 

 almonds which cleaves this coenzyme in a fashion such that the products 

 are the labile nicotinamide nucleoside, adenosine, and phosphoric acid. 25 



Since the coenzymes cannot be prepared synthetically, they must be 

 isolated from natural sources. Yeast is a rich source from which the 

 diphospho derivative is usually isolated, although it has been suggested 

 that better preparations can be obtained if muscle is used as the source. 

 Several investigators have reported alterations in procedure for improv- 

 ing the older methods of concentration and purification. 26 ' 21 Erythro- 

 cytes were the source originally used for preparing the triphospho com- 

 pound; 15 it has also been isolated from liver 28 and yeast. 29 The diphospho 

 nucleotide can now be purchased from commercial sources, and prepara- 

 tions possessing triphospho nucleotide activity can be prepared chem- 

 ically from the former coenzyme. 30 Although in their chemical properties 

 the di- and triphospho nucleotides are very similar, there are sufficient 

 differences in their tendencies to be absorbed 29 and in the solubilities of 

 their salts that their separation from one another is not difficult. 29 - 31 - 32 



The dihydro derivatives can be readily prepared by chemical reduction 

 of the oxidized forms of the coenzymes. Sodium hydrosulfite is a con- 

 venient reducing agent for this transformation. 33 



Assay Methods. The quantitative determination of the coenzymes of 

 nicotinic acid has been carried out by spectographic measurements, 

 enzymatic analyses, microbiological assays, and chemical methods. 



Both the characteristic absorption band at 340 m/x 27 and the fluores- 

 cence of the dihydrocompounds 21 have been employed for estimating the 

 coenzyme content of concentrates. These methods do not distinguish 

 between the di- and triphospho derivatives of nicotinic acid, but they 

 do serve as a means of establishing the combined amounts of the reduced 

 forms of the two coenzymes. 



The only specific method for determining the concentration of the 

 individual coenzymes is by use of enzymatic systems. With an appro- 

 priate apoenzyme an assay for either the diphospho or the triphospho 

 nucleotide can be accomplished. In this case both the oxidized and reduced 

 states of the particular coenzyme are equally active and are not dis- 

 tinguished by the method. In the determination of diphosphopyridine 

 nucleotide care must be taken to have an apoenzyme preparation free 

 from phosphatases which would degrade any triphospho coenzyme present 

 into the compound being measured. 



