362 RIBOFLAVIN 



tion by fluorometry are: stability in acid solution, adsorption on fuller's 

 earth from dilute acid, elution from fuller's earth by dilute pyridine, sta- 

 bility to weak oxidizing agents, reversible reduction to the leuco form by 

 hydrosulfite, destruction by visible light or ultraviolet light, transforma- 

 tion to lumiflavin by irradiation in alkaline solution, insolubility in CHCI3 

 whereas lumiflavin is soluble in this solvent, solubility in benzyl alcohol 

 and butanol-pyridine mixtures, and a characteristic fluorescence which is 

 optimum between pH 6 and 7 and completely depressed in strong acid or 

 alkali. The object of the various methods is to effect a sufficient separation 

 from other material so that the fluorescence measured is that of riboflavin 

 alone. 



a. Methods Utilizing the Fluorescence of Riboflavin 



Practically all the modifications of the fluorometric method in common 

 use have been described in detail by Stiller^ and Jones. ^ The completeness 

 of these reports makes any extensive description of the technical details 

 unnecessary at this time and allows less reference to the original literature 

 than would otherwise be necessary. Readers are referred to these presenta- 

 tions for a description of the methods. It appears more useful to direct this 

 discussion toward an evaluation of the methods available in so far as this 

 is possible. 



The principle steps of the fluorometric analysis include extraction of the 

 sample, removal of interfering materials, and measurement of the ribo- 

 flavin in the resultant extract. Reference to standard amounts of riboflavin 

 and correction for the fluorescence of suitable blanks must be included in 

 the latter step. 



(1) Extraction. The usual procedure is a hot dilute acid extraction, or 

 enzymatic hydrolysis, or both. The acid is ordinarily between 0.04 and 

 0.25 N. Several acids have been used, such as H2SO4 , HCl, and H3PO4 , at 

 either boihng temperature or in the autoclave. The enzymatic treatment 

 may be principally the action of phosphatases or nucleotidases (takadia- 

 stase, clarase, mylase, etc.) to free riboflavin from the nucleotides, or it 

 may also include a proteolytic enzyme, usually papain, to liberate ribo- 

 flavin bound to proteins. Since the fluorescence of flavin adenine dinucleo- 

 tide is relatively much less than that of free riboflavin' • ^ and much of the 

 riboflavin may appear in this form^"^ the desirability of sufficient hydrolysis 



1 E. T. Stiller, in Vitamin Methods, Vol. I. Academic Press, New York, 1950. 



2 J. H. Jones, in Vitamin Methods, Vol. II. Academic Press, New York, 1951. 



3 H. B. Burch, O. A. Bessey, and O. H. Lowry, /. Biol. Chem. 175, 457 (1948). 

 ' G. Weber, Biochem. J. 47, 114 (1950). 



6 J. L. Crammer, Nature 161, 349 (1948). 



« 0. A. Bessey, O. H. Lowry, and R. H. Love, /. Biol. Chem. 180, 755 (1949). 



7 K. Yagi, J. Biochem. (Japan) 38, 161 (1951). 



