162 A SYMPOSIUM ON RESPIRATORY ENZYMES 



activity toward methylene blue and oxidase activity toward 

 p-phenylenediamine or hydroquinone. It lacked, however, the ca- 

 pacity to oxidize succinate, apparently being unable to reduce 

 cytochrome c. When supernatant fluid from the ultracentrifuge run 

 was added, aerobic activity of the preparation toward succinate was 

 restored. The unknown material is evidently a substance of lower 

 molecular weight (estimated at 140,000), is heat-labile, and is re- 

 moved by trichloracetic acid, hence is probably a protein. It can- 

 not be identified with aluminum (61), catalase, or the Straub flavo- 

 protein (55). 



Recently Keilin and Hartree (9) have tested and analyzed the 

 eflFects of several factors on succinate and p-phenylenediamine oxi- 

 dation by typical heart muscle extracts. Among their findings were 

 these: 1. Narcotics inhibited the oxidation of succinate by the cyto- 

 chrome system more strongly than the oxidation of methylene blue. 

 Since not only reduction of the cytochrome components was in- 

 hibited, but also oxidation of cytochrome h, it is possible that these 

 findings are related to the function of cytochrome h in succinate 

 oxidation. 2. Preparations treated with alcohol modified irreversibly 

 the spectrum of cytochromes a^, a, and h and destroyed oxidase 

 activity. Such preparations did not reduce cytochrome c, although 

 they retained their ability to reduce methylene blue. 3. Treatment 

 with acetic acid (pH 5.0 for one hour) did not affect p-phenylene- 

 diamine oxidation, but destroyed the ability of the succinate system 

 to reduce cytochrome c. Again, methylene blue reduction was still 

 possible. Spectroscopically, the absorption bands of the cytochromes 

 were normal, except that cytochrome h appeared to be no longer 

 autoxidizable. Evidently cytochrome h had undergone some change. 

 4. Treatment with pancreatin gave a preparation similar to the 

 acid-treated preparation. 



The results suggest that cytochrome h may be the labile link 

 between succinic dehydrogenase and cytochrome c, although the 

 possibility that a flavin or another hematin is a link is by no means 

 excluded. It may be recalled that the potential of cytochrome h 

 (—0.04 V.) places it in a favorable position as such a link. Keilin 

 and Hartree have suggested as an alternative the possibility that 

 the failure to react with cytochrome c "may be due to an irreversible 

 change in the colloidal structure of the preparation accompanied 

 by a loss of accessibility of the succinic system to c, which is a non- 

 diffusible protein while it remains still accesible to small and dif- 

 fusible molecules of methylene blue." 



