276 



Discussion 



transport down the intact respiratory chain by rather high concentrations of cyto- 

 chrome c; here there is apparently no problem of availability of the oxidase to the 

 reaction site of cytochrome c. 



Henderson: I wish to ask Lucile Smith whether the result was the same, independent of 

 the iron concentration of the cytochromes ? We have found that both the preparations 

 with 0-34% and 0-43% iron, but particularly that with low iron, combine adequately 

 with copper. This is interesting in view of Wainio's paper. In the low-iron prepar- 

 ation, the cytochrome c is abready combined with non-basic proteins. Did you find 

 any effect of long incubation time as observed by Tsou in 1951? He regained the 

 complete endogenous activity on long incubation. 



Smith: We used cytochrome c of 0-34% iron, and the same preparation purified according 

 to Margoliash, with identical results. In our hands the period of incubation makes 

 no difference. 



Interaction of Cytochrome c with Other Compounds 



The Effect of Cations on the Reactivity of Cytochrome c in 

 Heart Muscle Preparations 



By R. W. EsTABROOK (Philadelphia) 



EsTABROOK : I would like to comment on a series of studies I have recently carried out 

 using a cytochrome-c-deficient heart muscle preparation in order to investigate the 

 role of cytochrome c in the succinoxidase system. These studies are complimentary 



180 



160 



120 



100 



60 



• 



004 0O8 012 0-16 



Phosphote buffer concenfrotion, mole/l 



Fig. 1. 



20 



to those of Smith and bring up a necessary question concerning the interpretation of 

 cytochrome c oxidase assays. During studies to assess the validity of a lipocytochrome 

 c as required for enzymic activity and to determine the accuracy of previous claims 

 that exogenous cytochrome c may be only 1/100 as active as endogenous cytochrome 

 c, it was observed that the K,n for exogenous cytochrome c, in the manometric 

 measurement of succinoxidase activity using the cytochrome-c-deficient heart muscle 

 preparation, was markedly affected by the presence of cations in the medium. This 

 is shown in Fig. 1 where the K^n is plotted as a function of the potassium phosphate 

 concentration employed in the reaction vessel. One sees over a 100-fold difference 



