604 B. Chance 



required to cause 50% inhibition (Chance and Hollunger, in preparation). 

 Studies on the DPNH oxidase system of a non-phosphorylating KeiHn and 

 Hartree heart-muscle preparation have shown that Amytal inhibits between 

 flavoprotein and the cytochrome chain, with an Amytal concentration of 

 0-16 mM required for 50% inhibition (Estabrook, unpublished data). This 

 modification of the locus of Amytal inhibition no doubt reflects the changes 

 in the nature of the electron transfer chain involved in the phosphorylating 

 and non-phosphorylating systems. 



Kinetic Studies of Respiratory Components 



Considerable interest is currently focused on determining both the reaction 

 sequence of electron transfer components in the Keilin and Hartree prepara- 

 tion and the interaction site of ubiquinone ; a closer examination of this 

 point is now made possible by the direct spectrophotometric recording of 

 ubiquinone reduction in collaboration with Dr. E. Redfearn. A comparison 

 of the results of rapid chemical assays with the spectrophotometric data 

 strongly supports the latter's applicability to the measurement of the kinetics 

 of ubiquinone. 



Summarized in Fig. 2 are four kinetic recordings representing the speed 

 with which cytochrome c, flavoprotein, cytochrome h, and an absorption 

 band in the ultra-violet, attributed to ubiquinone, change their intensities 

 upon adding 4 mM succinate to a cyanide-inhibited (1-2 mM) Keilin and 

 Hartree heart-muscle preparation. In records A and C, a downward deflec- 

 tion signifies an increase of absorbancy at the measuring wavelengths, 550 

 and 562 m//, corresponding to the reduction of cytochrome c and cytochrome 

 b, respectively. In records B and D, a downward deflection represents 

 decreased absorbancy at the measuring wavelengths, 465 and 275 m/^, and 

 thus the reduction of oxidized flavin and of oxidized ubiquinone. In view 

 of the fact that the protein absorption interferes with the measurement of 

 ubiquinone reduction, the reaction kinetics of this component are measured 

 with a 63-fold dilution of the heart-muscle preparation while those of 

 cytochrome b, cytochrome c, and flavin are measured with a 13-5-fold 

 dilution. The results are, however, comparable if the dilution factors are 

 taken into account. The kinetics are evaluated in terms of the initial slope 

 of the traces which, in the case of cytochrome c (record A), is about as rapid 

 as can be measured with the mixing technique employed; thus the initial 

 slope of 2 /^moles of Fe l.~^ sec"^ is an approximate value. In the case of 

 flavin, cytochrome b, and ubiquinone, the initial slopes can be obtained with 

 better accuracy. (The gaps in the traces for flavin and ubiquinone result from 

 the stirring interval.) The results of these kinetics, all calculated to a 63-fold 

 dilution and including additional data measured in the presence of antimycin- 

 A, are summarized in Equation 5, where the numbers along the arrows 

 represent the reduction rates of the component to the right of the arrow. 



