VII. ESTIMATION 3G9 



A i)i)lari^iaphi(' inctlunl was ilesciihcd by Liii<!;aiio and l)a\i(l.'' 'I'liis 

 method also apjiears less sensitix'c Ihaii those in common use and has had 

 very little application. 



3. Enzymatic Methods 



In their paper on the isolation of the prosthetic group of D-amino acid 

 o.xidase, flavin adenine dinucleotide, Warburg and Christian'" describe an 

 assay method for this riboflavin derivative. This consists in the measure- 

 ment of oxygen uptake when the material to be assayed is added to an 

 appropriate buffered solution containing the protein moiety of the enzyme 

 and DL-alanine as the substrate. The method has been used by various 

 groups^^'^^ and was modified to measure the apoenzyme instead of the 

 prosthetic group by Xegelein and Bromel.'*" Recently Comline and What- 

 ley"*^ have used the method to study the destruction of FAD by an en- 

 zyme in animal tissues, particularly in spleen. 



The method used b}^ Kuhn and Rudy^^ for the estimation of the prosthe- 

 tic group of the yellow enzyme, riboflavin mononucleotide, appears to have 

 had little use. This also utilizes oxygen uptake but with hexose phosphate 

 as the substrate, the specific protein of the yellow enzyme, and appropriate 

 coenzymes to complete the oxidizing system. Oxygen uptake is a function 

 of the amount of riboflavin phosphate added. 



B. BIOLOGICAL METHODS 



D. M. HEGSTED 



The Bourquin and Sherman^^ rat assay was the first method for the esti- 

 mation of what was then called vitamin B2 or G to receive rather wide ac- 

 ceptance. When pure riboflavin became available, it was shown that this 

 method gave quantitative responses to graded doses of riboflavin within 

 certain limits of growth. Although biological assays have since been im- 

 proved, the early development of the microbiological assaj'^ apparentl^y satis- 

 fied most of the requirements for a biological method, and the riboflavin 

 assay has never been subjected to the study and refinement which some of 

 the other vitamin assays received. The perfect assay diet would be one in 

 which all the nutrients required by the assay organism, with the exception 



« J. J. Lingane and O. L. Davis, /. Biol. Chem. 137, 567 1941). 



" O. Warburg and W. Christian, Biochem. Z. 298, 150 (1938). 



" S. Ochoa and R. J. Rossiter, Biochem. J. 33, 2008 (1939). 



^« J. R. Klein and H. I. Kohn, J. Biol. Chem. 136, 177 (1940). 



« E. Negelein and H. Bromel, Biochem. Z. 300, 225 (1939). 



^8 R. S. Comline and F. R. Whatley, Nature 161, 350 (1948). 



« R. Kuhn and H. Rudy, Ber. 69, 2557 (1936). 



6« A. Bourquin and H. C. Sherman, /. Am. Chem. Soc. 53, 3501 (1931). 



