580 E. C. Slater and J. P. Colpa-Bcx)nstra 



method does not require the determination of the total cytochrome b content. 

 The redox potential of cytochrome b was calculated from these values of K 

 and the potential of the succinate/fumarate system, +24 mV at pH 7, 25°C 

 (Borsook and Schott, 1931). The results ranged from +60 mV to +90 mV, 

 with a mean value of +77 mV. These values obtained both from the y- and 

 the a-bands agree very closely with the data for the a-region published by 

 Chance (1958), which would give an oxidation-reduction potential of 73-76 

 mV. However, Chance (1958) does not calculate a potential, because the 

 directly determined equilibrium constant (42-54) did not agree with the 

 ratio (10) of the second-order reaction constants for the forward and back 

 reactions. It seems to us that when the reduction has reached a steady state 

 in the absence of oxygen (when there can be no net electron transfer) the 

 system has reached equihbrium : and that the disagreement between the two 

 values must be ascribed either to experimental errors, or to the fact that the 

 full expression for the equilibrium constant probably contains rate constants, 

 which are not included in the bimolecular rate constants because they are not 

 rate-limiting. The reduction of cytochrome b by succinate is perhaps rather 

 complicated* (see below). 



The difference between the 77 mV found by Colpa-Boonstra and Holton 

 (1959) and the -40 mV of Ball (1938) is partly accounted for by the fact 

 that the latter assumed that all the cytochrome b reducible by Na2S204 could 

 also be reduced by succinate. This is not the case (Chance, 1952, 1958; 

 also, see below). 



KINETICS OF THE REDUCTION OF CYTOCHROME b 

 We have measured the kinetics of the reduction of cytochrome b in the 

 heart-muscle preparation by the two methods introduced by Chance (1952); 

 (i) by following the process of reduction from the aerobic steady state to the 

 anaerobic state while oxygen is consumed in the oxidation of succinate or 

 DPNH ; (ii) by measuring the rate of reduction by succinate in the presence 

 of cyanide. 



Figure 2, in which the recorder traces obtained with Holton's (1957) 

 instrument have been converted into absorbancy changes, shows that the 

 reduction of cytochrome b (measured at 432 m/^) and of cytochrome a^ (at 

 448 mfji) by succinate or by DPNH, as the system proceeds from the aerobic 

 steady state to the anaerobic state, are closely synchronized. Cytochrome b 

 was followed at 432 m^, because at this wavelength reduction of this cyto- 

 chrome results in a large increase in absorbancy, while reduction of the other 

 cytochromes causes a decrease. If the reduction of cytochrome b occurred 

 much later than that of cytochrome ^3 this would be revealed by a small 

 decrease of the absorption at 432 m/<, at the same time as the rapid increase 



* Added in proof . Another explanation of this discrepancy has been given in the full paper 

 by Holton and Colpa-Boonstra {Biochem. J. 76, 179, 1960). 



