98 Britton Chance 



substrate (Chance and Williams, 1955a). Approximately 

 25 [iM ADP gives half-maximal respiratory stimulation. Two 

 previous estimates of the ADP affinity of the respiratory 

 chain have appeared in the work of Slater and Holton (1953). 

 Their direct measurement gave a value of 7 X 10"'^ which 

 disagrees strongly with the results obtained here. Another 

 value of 10~^, obtained by a rather indirect estimation of the 

 affinity of isolated mitochondria for endogenous ADP, is 

 nevertheless within the range of values found here. 



Cytochrome <7 plus I84;jM Azide 605-630rnp log Io/I=0.003 



.±. 



74JJMADP 205uMADP ^ 20 



37;jMADP ^^J^^^^^ i^ ^ ^ sec. 



Fig. 4. Cyclic responses of cytochrome a of azide-treated guinea-pig 

 liver mitochondria obtained in response to increasing concentrations of 

 ADP. The separate additions of ADP are made to the same preparation 

 after the effects of the preceding ones have gone to completion. (Expt. no. 

 388c) (From Chance and Williams, 1956a. Reproduced by permission 

 of the Editors, J. biol. Chem.) 



Spectroscopic measurement of the magnitude of effect of 

 added ADP or phosphate upon the steady-state oxidation- 

 reduction level of the respiratory carriers provides a much 

 more sensitive method of determining the ADP affinity of the 

 respiratory carriers themselves (Chance and Williams, 19556). 

 In Fig. 4, the mitochondria are diluted in a medium containing 

 substrate and phosphate and are treated with a small con- 

 centration of azide so that a more distinctive response of 

 cytochrome a to ADP addition can be obtained (Chance and 

 Williams, 1956a). Thus, the addition of a small concentration 

 of ADP (37 (jlm) causes a small cycle of reduction of cytochrome 

 a, followed by its oxidation when the added ADP is expended. 



