io6 



THOMAS P. SINGER 



reaction sites in the DPNH-oxidase chain of heart mitochondria, the 

 flavoprotein and cytochrome c, just as in the succinoxidase system [13, 14]. 

 The relatively flat part of the curve represents primarily the reaction with 

 cytochrome c, since the apparent Kj^j of the flavoprotein for ferricyanide 

 is much larger than that of the cytochrome c site and thus, at low dye 

 concentrations, the measured rate represents largely the reaction with 

 cytochrome c. That this is the case may be readily shown by the following 

 facts : the flatter curve is non-competitively inhibited by amytal or antimy- 

 cin A (open circles), as expected from the fact that these inhibitors prevent 

 the flux of electrons to cytochrome c. It is competitively activated by cyanide 

 and azide (closed circles), because these inhibitors inhibit the flux of 

 electrons via cytochrome oxidase to Oo and thereby increase the flux of 

 electrons to ferricyanide. The activation is competitive with respect to 



Antimycin C x) y^ 



Amytal (o) ^^^^ ^■^ 



x/>* ^ ' Control 



4C*'^ +CN- 



10 



12 



2 4 6 8 



I/ml. FeCCN)^ ** 



Fig. 2. Ferricyanide assay of DPNH oxidation by ETP in the presence of 

 2 X io~* M DPNH. Conditions as in Fig. i. 



ferricyanide, since at infinite ferricyanide concentration all the electrons 

 would flow to the dye. The steep part of the curve represents the sum of 

 both reaction sites of ferricyanide. When the values from the extrapolation 

 of the flat curve are subtracted from the experimental values obtained at 

 high ferricyanide concentrations, the inhibition by antimycin and. amytal 

 and the activation by cyanide or azide disappear in accord with the fact 

 that these inhibitors do not afl"ect the DPNH-flavoprotein-ferricyanide 

 reaction sequence. 



Attention should be called to the fact that the higher the concentration 

 of ferricyanide employed, the greater the contribution of the dehydro- 

 genase site to the measured activity. Thus it is clearly desirable to employ 

 as high a concentration of the electron acceptor as compatible with the 

 optical arrangement when working at fixed concentrations of the oxidant. 

 In heart particles high concentrations of ferricyanide do not appear to be 



