144 THE BIOCHEMISTRY OF B VITAMINS 



Assay Methods. A D-amino acid oxidase system is the one commonly 

 employed for the qualitative and quantitative determination of flavin 

 adenine dinucleotide. 63 No other naturally occurring flavin derivative is 

 known to exhibit appreciable activity in this assay ; hence, it is used as a 

 specific test for the dinucleotide coenzyme. The apoenzyme concentrate 

 used in the assay is obtained by resolving the enzyme present in crude 

 extracts prepared from hog kidney. As in most flavoproteins, the pigment 

 dissociates from the protein when a solution of the holoenzyme is acidified, 

 and the protein component can usually be separated by ammonium sulfate 

 precipitation. D-alanine is the substrate most often used with this sytsem, 

 and oxygen is used as the hydrogen acceptor, since methylene blue is 

 reduced very slowly. The rate of the reaction is followed by measuring 

 the oxygen uptake or by determining quantitatively either the amount 

 of ammonia or of the keto acid formed by the oxidation of the amino acid. 



No enzymatic method has been developed to determine specifically 

 riboflavin phosphate. The use of the "old yellow enzyme" — the system 

 in which riboflavin phosphate was first recognized — will not suffice, since 

 the system is quite nonspecific in regard to its coenzyme requirements. 

 The dinucleotide, as well as certain riboflavin derivatives prepared syn- 

 thetically, can reactivate the apoenzyme (p. 150) . Cytochrome reductase 

 or L-amino acid oxidase, the only other enzyme systems whose coenzymes 

 are definitely known to be riboflavin phosphate, may be used. Whether 

 these systems are specific or not, however, is uncertain since no report has 

 been made concerning the activity of the dinucleotide when tested with 

 the apoenzymes of these proteins. 



Occurrence. Qualitatively, it is known that the coenzymes of ribo- 

 flavin have a widespread distribution in cells and tissues. The presence 

 of flavoenzymes (especially the dinucleotide type) has been demonstrated 

 in a variety of material, and the coenzymes themselves have been con- 

 centrated and isolated from microorganisms and several different animal 

 tissues. Much of the intracellular riboflavin is in a bound form from 

 which it can be liberated by phosphatases. If riboflavin resembles the 

 other B vitamins in respect to the distribution of the free vitamin and 

 its coenzymes, one would expect the intracellular vitamin content to 

 reflect predominantly the concentration of the coenzymes. However, prac- 

 tically no quantitative data, based on actual determination of the coen- 

 zymes, are yet available, nor is it possible to estimate the relative con- 

 centrations of the two coenzymes. 



Biosynthesis. The biosynthesis of riboflavin phosphate from the vita- 

 min has been carried out in vitro by using phosphorylating enzymes of 

 the intestinal mucosa of mammals. 64 The biosynthesis of the dinucleotide 

 has never been observed except in intact cells. Since this coenzyme cannot 



