HEMOGLOBIN AS A CYTOCHROME OXIDASE MODEL 391 



benzene is reduced back to phenylhydroxylamine by reducing systems 

 present in the erythrocyte (c/. Chapter XI). 



There is, however, more recent evidence confirming the existence 

 of different hemiglobins. Darling and Roughton {532) have observed 

 differences between the hemzglobin produced by ferricyanide and 

 that produced bj' autoxidation in their effect on the dissociation 

 curves of oxyhemoglobin. An alteration of the globin in one of the 

 reactions leading to hemoglobin was suggested as an explanation. 

 Vestling {2873) found that hem/globin formed with nitrite is reduced 

 much more slowly and less completely by ascorbic acid at pH 7 and 

 0° C. than the hemiglobin formed with ferricyanide. This was 

 assumed to be due to a difference of the two hemiglobins. Doubt 

 was cast on this explanation by Gibson {993), since the addition of 

 ferrocyanide to the nitrite-hem/globin -ascorbic acid system in- 

 creased the rate of reduction to that found for ferricyanide hemo- 

 globin. The author himself, however, points out that it is impossible 

 to assume that ferrocyanide reacts by reducing the ferric iron of 

 hemiglobin directly. The catalytic effect of ferrocyanide thus remains 

 unexplained. 



6.3.4. Formation of Hemiglobin by Reducing Substances in 

 the Presence of Oxygen. A number of dyes oxidize hemoglobin to 

 hemiglobin in the presence of oxygen, but it is clear from a consider- 

 ation of the oxidation-reduction potentials that only a few {e.g., 

 phenolindophenol with a potential of + 0.250 v. at pH 7) oxidize 

 hemoglobin (potential + 0.150 v.) directly. The most important of 

 the hemtglobin-forming dyes and substances, however, have a poten- 

 tial considerably below that of hemoglobin, e.g., methylene blue 

 (potential + 0.010 v. at pH 7.0); dyes of a still lower potential fail 

 to form hemiglobin {191^1). 



It has been assumed that methylene blue, for example, catalyzes 

 hemiglobin formation by oxidizing hemoglobin and by being con- 

 stantly re-formed from leucomethylene blue by autoxidation. 

 Although the oxidation reduction potentials of methylene blue- 

 leucomethylene blue and hemiglobin -hemoglobin show that the reac- 

 tion methylene blue + hemoglobin —* leucomethylene blue -f hemi- 

 globin can each time proceed only very slightly toward the right-hand 

 side, it is theoretically not impossible that a catalysis of hemiglobin 

 formation may be brought about in this manner (c/. Michaelis and 

 Salomon, 19Itl; De Meio, Kissin, and Barron, 552). 



