540 



H. Hasegawa and Y. Ogura 



a linear function of the dye concentration, while it became saturated at higher 

 dye concentrations. It was further checked that at these saturating concentra- 

 tions of dyes, dye concentrations decreased linearly with the reaction time. 



30 



o 2-0 



X 



10 



1-0 

 l/[s] 



X I0-* (M-i) 



2-0 



Fig. 6. Relationships between the reciprocal of rate and the reciprocal of substrate 



concentration. Hydrogen acceptor, thionine. Experiments were carried out at 



pH 7-2. 



Using various kinds of dyes, the dependency of the rate upon the concentra- 

 tion of substrate was investigated according to the method of Lineweaver and 

 Burk (1939). 



The data obtained are illustrated in Fig. 6. The maximum rate/unit concen- 

 tration of the enzyme {y\e) and the Michaelis constant (A',„) are listed in 

 Tables 1 and 2. The values of Vie as well as those of A',,^ appeared, within the 

 experimental error, to be the same with the different hydrogen acceptors used. 

 It was also found by the double reciprocal method reported by Singer and 

 Kearney (1957) that the same situation holds at lower concentrations of the 

 dye; the result was essentially the same as that given in Table 1. 



The oxidation of reduced cytochrome h,^ by ferricyanide and thionine was 

 demonstrated by spectrophotometric observations. In tiie experiments repro- 

 duced in Figs. 7 and 8, the oxidation of cytochrome b.^ proceeded to com- 

 pletion with an excess of ferricyanide (Fig. 7), but only to a certain extent on 



