Action of Yeast Lactate Dehydrogenase 



537 



to the reduction of cytochrome b^ as was first assumed by Appleby and 

 Morton (1954). 



To elucidate further the situation of electron transfer in the enzymic 

 reaction, spectrophotometric measurement was made of the oxidation- 

 reduction potential of cytochrome b^. An enzyme solution was placed in a 



240 



sec 

 Fig. 2. Inhibitory effect of riboflavin on the enzymic reaction. Concentrations 

 of riboflavin were 200 //m (curve 1), 100 /<m (curve 2), 32 /<M (curve 3), 10 fiM 

 (curve 4), 3-2 //m (curve 5), respectively. Curve 6 represents the control. Experi- 

 ments were carried out at 25''C and pH 7-2. 



Thunberg tube, the lower part of which was shaped like an optical cell, and 

 which was fitted with a rubber stopper. The side-bulb of the tube contained 

 a solution of reduced toluylene blue (TB) (Eq = 0-127 V, pH 7-0) which was 

 to be used as an indicator. After the air in the vessel was completely replaced 

 with nitrogen gas, about half of the toluylene blue was reduced by dithionite 

 using a syringe through the rubber stopper. The mixture of both oxidized 

 and reduced forms of dye was introduced into the buffered enzyme solution. 

 By measurement of the changes of optical density at 557 and 600 m/u, the 

 oxidation-reduction potential of cytochrome b^ could be determined accord- 

 ing to the following equation : 



