MECHANISM OF CHOLEGLOBIN FORMATION 475 



of the absorption spectra. Also, denatured globin or pyridine chole- 

 hemochrome does not yield azahemochrome with ammonia.* The 

 similar mode of its preparation, the lack of the Soret band, the ease 

 with which iron is detached, and the fact that the same bile pigments 

 (biliverdin and biliviolinoid substances) are obtained in this way, 

 however, all indicate a close relationship. 



If choleglobin or "green pigment" is incubated for sixteen hours at 

 37° in 66% acetic acid, two thirds of its iron is removed. The bile 

 pigment yields from choleglobin are small, however, only about 10% 

 of the theoretical. Very little bile pigment is obtained from "green 

 pigment," the prosthetic group remaining firmly attached to the 

 denatured protein, in spite of the removal of iron. 



From the fact that a constant proportion of choleglobin is transformed to 

 bile pigment, and from the constant ratio, + ACesomp/— Aeo7om,i, Lemberg 

 ef al. concluded that the bile pigments are derived from chole- 

 globin by the action of the acetic acid. It is not impossible, however, that 

 choleglobin is a precursor of verdohemoglobin, and that the latter is unstable 

 under the conditions of the experiment and breaks down to yield free bili- 

 verdin. There are some indications for this. Compounds similar to verdo- 

 hemochrome, although apparently not identical with it, were obtained by 

 coupled oxidation of "green pigment" with ascorbic acid in 20% pyridine, 

 and also during the late stages of the coupled oxidation of hemoglobin with 

 ascorbic acid. By splitting of green pigment with acid, a very small amount 

 of an oxyporphyrin-like substance was obtained in addition to bile pigments; 

 this compound could not, however, be obtained from choleglobin solutions 

 (1709). Finally, the ratio Ceso m^/fesomM is 0.6 for choleglobin — higher 

 than that for "green pigment" (0.32) — which might indicate an admixture 

 of a verdohematin derivative in the former. If the ratio of choleglobin to 

 verdohemoglobin formations remains constant during the initial stages of the 

 action of ascorbic acid, a small amount of verdohemoglobin formation may 

 be difficult to detect spectroscopically. 



The problem of the constitution of the prosthetic group of choleglobin 

 must remain open. 



4.4. Mechanism of Choleglobin Formation 



4.4.1. Principle of the Mechanism. Hydrogen peroxide acting 



on hemoglobin in the presence of ascorbic acid produces choleglobin 



{1708). Even in the absence of ascorbic acid, hydrogen peroxide 



transforms hemoglobin, and also to a smaller extent oxyhemoglobin 



and hem/globin, into choleglobin {1710). 



* Liebecq {1738c) has recently claimed that choleglobin as well as pseudohemoglobin 

 yields azahemochrome with ammonia; a small azahemochrome band appeared in 

 ammonia, but the band in the orange of choleglobin or pseudohemoglobin did not 

 disappear. 



