HUGO THEORELL 



the fluorescence of riboflavin disappeared in alkaline solution, 

 and that the disappearance followed a dissociation curve with 

 pK.' = 10.2 and n = 1. The corresponding acid group was 



considered to be the imino group NH in the 3-position, and in 



analogy with the quenching of the fluorescence by the dissocia- 

 tion of a proton from this group, Kuhn and Boulanger (6) ex- 

 plained the absence of fluorescence in the O.Y.E. by a coupling 

 of this imino group to an unknown group in the protein. 



In 1937 (18), Theorell used the disappearance of the 

 fluorescence of FMN upon recombination with the apoprotein 

 as the basis for "titrating" this protein with solutions of FMN. 



Since nowadays very sensitive photomultipliers and rapid 

 recorders are available, we recently decided to study the ki- 

 netics of the dissociation and reassociation of the O.Y.E. by the 

 aid of fluorescence measurements (26). No doubt it would have 

 been technically possible to carry out such studies spectro- 

 photometrically with the aid of the rapid-flow technique, since 

 the absorption band at 445 m/x of free FMN shifts to 465 m/x 

 in the O.Y.E. Such combination experiments had been 

 carried out some years ago by Theorell and Chance (24) in the 

 case of DPNH + alcohol dehydrogenase (ADH), where the 

 absorption band of DPNH at 340 m/i shifts to 325 m/x when 

 DPNH is combined with ADH. 



In the case of the O.Y.E. we actually found the same ki by 

 the aid of a recording spectrophotometer, at 495 m^u, and by 

 fluorescence measurements on the same solutions. The ex- 

 periments had to be carried out in concentrations high enough to 

 give enough light absorption, and with high concentrations of 

 phosphate to reduce the rate of the association reaction to 

 measurable dimensions. 



It was thus found that the fluorescence method has some 

 distinct advantages over spectrophotometrical methods. The 

 reason is that spectrophotometry is practically possible only 

 down to concentrations around lO^'^Af, whereas by the aid of 



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