536 



H. Hasegawa and Y. Ogura 



follows. After the enzyme solutions were mixed with various concentrations 

 of riboflavin, the rate of reduction of thionine was measured (by the extinc- 

 tion at 660 m/i) in the presence of an excess of substrate. As may be seen 

 from the results presented in Fig. 2, the inhibition caused by riboflavin 

 increased with time as the reaction proceeded. It is worth mentioning that 

 no inhibitory eff"ects of riboflavin could be observed when the rate of reduction 

 of cytochrome b^ was measured under similar conditions. The data presented 

 in Fig. 2 seem to suggest that the flavin mononucleotide bound to the enzyme 



0-2 



o 



400 



450 



Wavelength, m// 



Fig. 1. Difference spectrum of flavin mononucleotide attached to the 

 enzyme protein. 



protein became mobilized and exchanged with the added riboflavin when the 

 enzyme was put into its function. 



It was further checked whether or not the flavin mononucleotide attached 

 to the enzyme could be removed on the addition of an excess of riboflavin. 

 After the enzyme solution was mixed with an excess of riboflavin, the mixture 

 was allowed to stand for several hours and then treated with ammonium 

 sulphate solution. The precipitate was washed several times with ammonium 

 sulphate solution. The flavin moiety of the enzyme thus treated was identi- 

 fied as riboflavin by paper chromatography. On adding the substrate to the 

 enzyme thus treated, the protohaem moiety of the enzyme was not reduced ; 

 the reduction could be achieved only by treatment with dithionite. The 

 observation made above indicates that the flavin mononucleotide moiety of 

 the enzyme can be replaced by riboflavin leading to the formation of an 

 inactive enzyme complex, and that the flavin mononucleotide attached to the 

 enzyme acts as the direct acceptor of electrons derived from substrate prior 



