134 THE BIOCHEMISTRY OF B VITAMINS 



genated when the pyridine nucleus is reduced has not yet been established. 



The reduction of the coenzymes not only changes the acidic and basic 

 properties of the compounds, but also causes characteristic alterations in 

 their absorption spectra, in their stability toward acidic and alkaline 

 treatment, and in their affinity for their apoenzymes. The reduced coen- 

 zymes both show in their absorption spectra a very distinct band at 

 320-360 m^ which is totally absent from the spectra of the oxidized 

 molecules. The production of this band during reduction of the coenzymes 

 is so characteristic of these compounds that it can be used as an analytical 

 method for determining the di- and triphospho nucleotides in purified 

 preparations. 19 Such spectrographs analysis also is an excellent method 

 for following the progress of enzymatic reductions or other enzymatic 

 reactions which can be coupled to a reaction involving a nicotinic acid 

 coenzyme. 20 When irradiated with ultraviolet light, only the reduced 

 coenzymes exhibit a strong whitish fluorescence. 21 



The coenzymes in their oxidized state are rapidly inactivated by stand- 

 ing at room temperature in dilute alkaline solution (0.1N), but the rate 

 of destruction is much slower in acidic solutions (O.liV) ; the reduced 

 molecules, on the other hand, are inactivated immediately by weakly 

 acidic conditions, but are unaffected by the alkaline treatment. 18 



The immediate destruction of the reduced molecules by acid has been 

 attributed to the formation of an addition compound in which a molecule 

 of acid adds to one of the double bonds of the dihydropyridine nucleus. 22 

 The stability of the dihydro coenzymes (trivalent nitrogen atom) in 

 alkaline solution is comparable to the stability of the glucosidic-like link- 

 age through the nitrogen atom of purine nucleosides (likewise trivalent) . 

 Hence, the unusual alkaline lability of the oxidized nicotinamide nucleo- 

 side can probably be attributed to the difference in properties of a glu- 

 cosidic bond linking a quaternary ammonium nitrogen ion to a pentose. 

 The initial cleavage occurring when diphosphopyridine nucleotide is sub- 

 jected to either acidic or basic hydrolysis takes place at the bond linking 

 the pyridine base to the ribose. 



Adenosine diphosphate and adenylic acid can be obtained from the 

 alkaline hydrolyzates of diphosphopyridine nucleotide, 23 the latter pos- 

 sessing the coenzymatic activity necessary for the activation of certain 

 apophosphorylases. Attempts to demonstrate (by this enzymatic anal- 

 ysis) the formation of adenylic acid by a comparable treatment of the 

 triphosphopyridine nucleotide have been unsuccessful. This is the reason 

 for believing that the third phosphate group of the latter coenzyme is 

 attached in the form of a phosphate ester of the adenosine portion of the 

 molecule rather than as a portion of the pyrimidine nucleoside or the 

 connecting pyrophosphate bridge. Alkaline cleavage of the former type 



