Studies on Problems of Cytochrome c Oxidase Assay 



263 



then against distilled water. Part of the salmine is lost through the Cellophane 

 membrane during dialysis. The final concentration of protein remaining 

 after dialysis was measured by the biuret method (Gornall, Bardawill and 

 David, 1949) and the molarity of the salmine calculated assuming a molecular 

 weight of 8000. 



RESULTS 



Effect of the Concentration of Cytochrome c on Cytochrome c Oxidase Activity 



Figure 1 shows semilogarithmic plots of the optical density minus the 

 optical density of totally oxidized cytochrome at the wavelengths indicated in 



001 



Time = ll-5sec 



Fig. 1. Semilogarithmic plots of the observed changes in optical density as ferro- 

 cytochrome c is oxidized by the oxidase. Ordinate values are E values at given 

 times minus E of the totally oxidized cytochrome c; abscissa represents time. 

 The different experiments were run with different concentrations of cytochrome c 

 in the tests, but with the same concentration of the oxidase. The data of curve 

 A were measured at 415 m/<; those of curves B, C, D, and E at 550 m/i, and 

 those of curve F at 520 m/<. 



A = 1-07 fiM cytochrome c 

 B = 5-33 /tM cytochrome c 

 C = 80 fiM cytochrome c 



D = 16 fiM cytochrome c 

 £■ = 32 ^fM cytochrome c 

 F = 128 /j,M cytochrome c 



the legend against time as ferrocytochrome c is oxidized by the oxidase. The 

 different hues were obtained with different concentrations of cytochrome c 

 in the test mixtures, the concentration of the oxidase being held constant. 

 From the slopes of the plots, the first order velocity constants can be calcu- 

 lated in each case; these are plotted against cytochrome c concentration in 



