RESPIRATION AND OXIDATIVE PHOSPHORYLATION 



117 



ni/t in the presence of this inhibitor. At the same time, at liquid ni- 

 trogen temperature, the Soret band at 444 m/x is split into three com- 

 ponents, an absorption maximum at 436 m^ and shoulders at 445 

 and 460 m/x. Assays on pure cytochrome oxidase by Yonetani and 

 Bonner (1961) confirm this finding and demonstrate a splitting of 

 the Soret band into two peaks at 436 and 445 ni/x and troughs at 442 

 and 450 m^. But azide action is apparently not confined to cyto- 

 chrome oxidase, as is exemplified by the results on steady state of 

 cytochrome b. 



When azide is added to preparations treated with dinitrophenol 

 and lactate, i.e., with high respiration, one again observes a further 

 reduction of cytochromes a and c and a net reduction, rather than 

 oxidation, of cytochrome b, contrary to the effect in the absence of 

 DNP (Fig. 10, dotted curve). The effect of azide on the cytochromes 



i 530 r 



II I.I. 



1610 mfj 



AO.D. 



0.005 cm 



Fig. 10. Difference spectra of bull spermatozoa at liquid nitrogen 

 temperature. Cells washed and suspended in RP, pH 7.2. Samples H4322, 

 H4722. Lower curves: solid line: (lactate 10 mM, aerobic) — (aerobic, 

 endogenous substrates); dashed line: (azide 10 mM, lactate, aerobic) — 

 (lactate, aerobic). Upper curves: solid line: (DNP 0.1 mM, lactate, aerobic) 

 — (lactate, aerobic); dashed line: (azide 10 mM, lactate, aerobic) — (lac- 

 tate, aerobic); dotted line: (azide, DNP, lactate) — (DNP, lactate). 



