ENZYME-SUBSTRATE COMPOUNDS 



their positions in the respiratory chain; for example, cyto- 

 chrome c is oxidized much more rapidly than flavoprotein. The 



Flow starts 



jl. Flow Velocity Trace 



550-540m>j T" 

 log lo/I = 0.010 



I5;jM 



Oxygen j^^ Cytochrome c 



oxidationt 



10 20 



Time after flow stops (sec) 



Flo w Velocity T r c e s 



-sei^ r^s y564-575m. rn'Lii ^65-510 m, 



/SbT632m>j Ioglo/I=a005 

 3 Io/I= 0.005 r^" "'~ 



Cytocfirome a Cytochrome b Flavoprotein 



Fig. 7. An experimental study of the time sequence of oxidation of four 

 members of the mitochondrial respiratory sequence. 



The top trace of each of the four records represents flow velocity, and 

 a downward deflection from the base line represents an increase of velocity. 

 Since the method involves both accelerated- and stopped-flow principles, the 

 interval of flow is only about V2 sec. The time after mixing corresponding to 

 the shortest time achieved in a particular record is marked on the figure. 



The spectrophotometric data are recorded by two monochromator 

 apparatus in which the optical density changes are recorded with respect to 

 a nearby neutral "reference" wavelength. The optical density changes at 

 the "active" wavelengths are registered so that a decrease of optical density 

 gives an upward deflection in all cases (the sense of deflections being the same 

 as in Fig. 5). Thus an oxidation gives an upward deflection in all cases 

 except flavoprotein. The complete cycle of oxidation and reduction is 

 shown for cytochrome c; only the initial phases of the reaction are shown for 

 the other three components. 



The speed of reaction is computed from the optical density change at- 

 tained at the highest value of flow velocity recorded by the flow velocity 

 trace. This value is converted into per cent completion of the oxidation 

 reaction by means of the maximum optical density change attained after 

 the flow stops. A summary of data is given in Table I (Expt. 367c). 



speeds of oxidation are more than adequate to account for the 

 observed respiration of these preparations. Any other re- 



331 



