478 X. BILE PIGMENT FORMATION, ETC. 



and are easily hemolyzed, e.g., by centrifugation. Still more unstable 

 choleglobin cells were prepared with phenylhj^drazine. The intra- 

 corpuscular formation of biliverdin by phenylhydrazine in vivo and 

 the small amount of biliverdin obtainable from normal erythrocytes 

 will be discussed in the next chapter. 



4.4.3. Green Pigment. Under the conditions of pH and tempera- 

 ture used for the coupled oxidation, hemoglobin itself (in the absence 

 of atmospheric oxygen) is not denatured. Even while the reaction 

 proceeds in air, at first only the choleglobin is denatured and pre- 

 cipitated; at this stage the green precipitate is alkali-soluble. Only 

 later is hemoglobin also denatured, the precipitate acquiring a less 

 purely green color and becoming insoluble in alkali. The denaturation 

 appears to be due to an oxidation of globin, to which choleglobin is 

 apparently more sensitive than hemoglobin. It is probably due to 

 hydrogen peroxide formed in the reaction. This oxidative denatura- 

 tion is accompanied by a change to an irreversible type of linkage 

 between prosthetic groups and protein, which we shall also find in 

 pseudohemoglobin^ cruoralbin (prepared at room temperature), and 

 sulfhemoglobin — in these cases without the protein becoming insol- 

 uble at neutral pH. For this reason the "green pigment" of Anderson 

 and Hart yields little ether-soluble substance on treatment with acid. 



4.4.4. Various Hydrogen Donors Producing Choleglobin. In addition to 

 ascorbic acid and ascorbic acid plus reduced glutathione, a variety of other 

 hydrogen donors produces choleglobin from hemoglobin. Reductone is quite 

 as active as ascorbic acid (Lemberg and co-workers, 1710). Adrenaline and 

 phenylhydrazine have been mentioned above. The hemoverdin of Lewin 

 was probably impure biliverdin. resulting from the treatment of choleglobin 

 with acid. A great variety of substances and systems was found by Anderson 

 and Hart {52) to form green pigment, e.g., dihydroxyacetone with ammonia 

 or glycine, glucose in phosphate with cysteine or glutathione, or with much 

 glycine, or with glycine and ammonia. 



Lemberg and co-workers {1710) found cysteine and glutathione alone 

 almost inactive, Barkan and Schales {163), only occasionally active. This 

 probably depends on the purity of the glutathione. With hydrogen peroxide, 

 cysteine and reduced glutathione have been found to yield green oxidation 

 products of hemoglobin {216Ji). Thioglycolic acid is not only able to form 

 choleglobin or a similar green pigment {163), but also to detach its iron 

 {25^7). Dimercaptopropanol rapidly reduces hemoglobin and causes coupled 

 oxidation {2968). 



Dialuric acid and alloxanthine as reducing substances form choleglobin, 

 while alloxan, an oxidizing substance, forms only hemoglobin {359). 



In the presence of oxygen, hydrogen sulfide acting on hemoglobin produces 

 choleglobin in addition to sulfhemoglobin, particularly if the reaction is 



