COENZYMES DERIVED FROM B VITAMINS 137 



all the nicotinic acid activity of a preparation exists in the form of the 

 coenzymes or if the various nicotinic acid-containing compounds are 

 separated by means of paper chromatography, for example. 



The use of any of these methods for the analysis of natural substances 

 depends upon obtaining extracts suitable for analysis. Considerable 

 variation can often be noted in the analytical results of different in- 

 vestigators who have tested crude materials. This would indicate that 

 the accuracy of these methods may be limited by the factors involved 

 in the preparation of the extracts. The instability of the coenzymes and 

 the enzymatic destruction which occurs when cells are macerated must 

 always be taken into account. In making differential assays for the two 

 coenzymes the possibility of enzymatic interconversion must be con- 

 sidered. The rapidity with which the original equilibrium of the oxidized 

 and reduced forms of the two coenzymes can be disturbed can lead to 

 erroneous conclusions when values for the two states of each of the 

 coenzyme are sought. The reduced coenzymes, for example, are oxidized 

 by air during hot-water extraction. 18 



Occurrence. In view of the number of essential reactions for which 

 the coenzymes of nicotinic acid are required, it is not surprising that they 

 have been found in all cells which have been examined for their presence. 

 It would be most unexpected if some type of cell were found which did 

 not contain these coenzymes. The quantities of the two individual enzymes 

 as well as the ratio between the di- and triphospho nucleotides vary over 

 a wide range for different types of cells. 18 Yeast, the richest source of the 

 diphospho nucleotide yet encountered, contains as high as 0.5 mg per 

 gram of moist cells. The triphospho nucleotide is always present in 

 smaller quantities than the simpler coenzyme, which would be expected 

 inasmuch as it functions in fewer reactions. 



The degree of association of these coenzymes with their apoenzymes 

 is less than that encountered in any of the other B vitamin coenzymes. 40 

 It has been pointed out that the coenzymes really function as substrates 

 of two independent reactions and that they are, in effect, catalysts for 

 a complex oxidation-reduction process rather than for simple individual 

 reactions. The coenzymes must alternate between two separate apo- 

 enzymes ; the one on which the coenzyme is reduced and the one on which 

 the reoxidized form is regenerated. Since comparable rates of reaction 

 are found in both intact cells and in solutions in which there is no organiza- 

 tion of the enzymes, it is believed that these two apoenzymes between 

 which the coenzyme shuttles need not be coupled together sterically 

 within the cell structure. 41 These coenzymes have consequently been 

 described as "mobile coenzymes" in order to contrast their behavior with 

 that of the fixed coenzymes which remain attached to one apoenzyme 



