538 



E'o (cyt b^) = £^(TB) + 0-03 log 



H, Hasegawa and Y. Ogura 

 [TB] 



[leuco TB] 



0-06 log 



[cyt b^ 



[cyt Z)2++] 



where E'^ (cyt b^ is the oxidation-reduction potential of cytochrome b^, and 

 E'q (TB) is the oxidation-reduction potential of toluylene blue. 



pH 



Fig. 3. Values of oxidation-reduction potential of 

 cytochrome b^ at various pH values. 



Figure 3 shows the relationships between the oxidation-reduction potential 

 of cytochrome b^, and the pH of the medium. The oxidation-reduction 

 potential of cytochrome ^2 thus obtained was + 0T2 V at 25^C and pH 6 to 8. 



Kinetics of the Enzymic Reaction 



To obtain information as to the mode of action of the enzyme some kinetic 

 investigations were performed. Figure 4 shows the relationships between the 

 rate of reaction and the concentration of enzyme in the presence of methylene 

 blue and ferricyanide as electron acceptors. As may be seen, the rate in- 

 creased proportionally with the enzyme concentration when both substrate 

 and dye were present in excess. The rates obtained from the slopes of the 

 curves were 1-3 x 10^ sec~^^ (methylene blue) and 1-5 X lO^sec^^ (ferri- 

 cyanide) respectively, the rate being defined as the amount of L-lactate 

 oxidized/unit concentration of enzyme/sec. The eflFect of dye concentration 

 upon the rate was investigated in the presence of an excess of substrate. 

 Figure 5 shows the relationships between the concentrations of dyes and the 

 rates at 25°C and pH 7-2. At lower concentrations of the dye, the rate was 



