RESPIRATORY FERMENT AND RELATED CYTOCHROMES 367 



cytochrome c by precipitation with an equal volume of 0.2 M acetate buffer 

 (pH 4.5) and resuspension in 0.1 M phosphate buffer, pH 7.4 {26S1). Stern 

 {2079, p. 264) found the enzyme in these preparations in relatively large 

 particles of remarkably homogeneous size.* The extracts with sodium cholate 

 and secondary phosphate have been mentioned above. Haas {107 Jf) obtained 

 a solution of cytochrome oxidase by grinding heart muscle with sand and 

 toluene, or by treatment with ultrasonic waves followed by extraction, 

 precipitation at pH 5.6, and redissolution at pH 9.5. All these solutions still 

 show the Tyndall phenomenon, but Haas's preparation was not thrown out 

 by short centrifugation at 12,000 g. In a later paper {1075) Haas described 

 the separation of the enzyme into two fractions, which have full catalytic 

 activity only in combination. The first is heat-labile and is precipitated on 

 prolonged (two hours') centrifugation at 10,000 g. The second is heat-stable, 

 slightly yellow, nondialyzable, and can be precipitated with ammonium 

 sulfate; redissolved, it loses its activity on standing. Addition of the latter 

 fraction to the former greatly increases its oxygen uptake. t 



Stotz and co-workers (2677) showed that cytochrome oxidase unites 

 with cytochrome c to form a complex for which the Michaelis-Menten 

 equation holds (Michaelis constant of 5.8 X 10^ M). By studying 

 the influence of increased cytochrome c concentration on the system 

 partly inhibited by cyanide or carbon monoxide, they confirmed that 

 in this complex the oxidase, not cytochrome c, reacts with the 

 inhibitors. Today the identity of cytochrome oxidase with the 

 respiratory ferment of Warburg is accepted by most workers. 



The claim of Japanese workers that the respiration of yeast and other 

 organisms is catalyzed by two different enzymes, one of which reacts with 

 carbon monoxide while the other reacts with cyanide, will be discussed in 

 Section 6.4. 



The estimation of cytochrome oxidase in tissues is not an easy task. It 

 has been reviewed by Potter {2177). The tissue is homogenized and the 

 concentration of the enzyme is measured in terms of oxygen uptake or 

 substrate disappearance in the presence of excess cytochrome c and substrate. 

 Several methods have been described {106,669,U7o,2o38,267If) . 



3.6.5. Cytochrome as. A spectroscopic investigation of cyto- 

 chrome oxidase preparations for a long time failed to give any results 

 indicating the presence of the absorption band at 589 mju which was 

 to be expected if cytochrome oxidase was identical or closely related 

 to Warburg's respiratory ferment {cf. 2677). In 1938 Keilin {H92) 

 found that on treatment of cytochrome oxidase preparations with 



* In rats' liver cells the oxidase is present exclusively in the mitochondria (Schneider, 

 Hogeboom, and co-workers, 21to2a). 



t According to Keilin and Hartree (1501a), the activation is, however, unspecific; 

 the soluble fraction can be replaced by plasma proteins or hematin-free denatured 

 globin. 



