RESPIRATION AND OXIDATIVE PHOSPHORYLATION 109 



voproteins, and pyridine nucleotides. No cytochrome c x , as found in 

 sea urchins (Keilin and Hartree, 1949), was detected. 



Concentration of Respiratory Enzymes 



From a number of spectra such as those shown in Figs. 1 and 2, 

 recorded at room temperature, one can calculate proportions and 

 concentrations of the respiratory enzymes. Concentrations were de- 

 termined under conditions where a difference spectrum would show 

 a maximum amount of reduced pigment. As noted above, this is 

 realized by recording the difference between the anaerobic suspen- 

 sion and an aerobic sample with very low endogenous respiration. 

 The respiratory inhibitor, Amytal, can also be used to oxidize pig- 

 ments in the reference sample. This compound interrupts electron 

 transfer between pyridine nucleotides and flavoproteins and thus 

 leads to the reoxidation of pigments located on the oxygen side of its 

 site of action, namely FP and cytochromes b, c, a, and a z . 



When Amytal is added to an aerobic suspension of starved bull 

 spermatozoa, even at this low respiratory level a small amount of 

 cytochromes b and c is reduced, approximating 10% of the total 

 amount shown in Table I. With dog and Spisula spermatozoa, 

 Amytal treatment of the reference sample proved to be an absolute 

 requirement. In the case of dog sperm only a light washing was ap- 

 plied to avoid possible loss of pigments, particularly of cytochrome 

 c. Respiration therefore remained high. The same situation pre- 

 vailed in Spisula where the endogenous respiration of spermatozoa 

 cannot be eliminated by repeated washing. With dog sperm 80% of 

 the endogenous respiration is Amytal-sensitive, and a rather com- 

 plete reoxidation of cytochromes may be expected, leading to rea- 

 sonably good figures for cytochrome concentration. In Spisula sperm, 

 however, as much as 50% of respiration (e.g., succinic acid oxida- 

 tion) is resistant to Amytal. For the terminal cytochromes a 3 , a, and 

 c, which are usually less reduced in the aerobic steady state, the values 

 in Table II may be considered correct. But values of cytochrome (b) 

 are probably low, as are those for flavoproteins. Data for pyridine 

 nucleotides are unreliable owing to the conditions of Amytal inhibi- 

 tion. 



As seen in Tables I and II, there is no significant difference among 

 the same bulls studied in different seasons or between dog and bull 



