MECHANISM OF CHOLEGLOBIN FORMATION 477 



ratio a/|8 is somewhat smaller than in the verdohemochrome forma- 

 tion, roughly 10 moles of ascorbic acid being oxidized per mole of 

 choleglobin formed. 



Hemoglobin is an intermediate of the cycle. By coupled oxidation 

 with ascorbic acid it is converted into choleglobin at the same rate 

 as oxyhemoglobin {1710). Cyanide, by transforming hemtglobin 

 into hemzglobin cyanide which ascorbic acid cannot reduce, inhibits 

 choleglobin formation (1315) ; by this it is shown that the process of 

 choleglobin formation is different from that of pseudohemoglobin 

 and cruoralbin formation for which a high concentration of cyanide 

 is required {cf. Section 6.). 



The reaction proceeds rapidly under physiological conditions of 

 pH, temperature, oxygen pressure, and concentrations of ascorbic 

 acid and reduced glutathione. At an oxygen pressure of 15 mm. of 

 mercury it is about four times faster than at 150 mm. pressure (1666, 

 1710); these optimal conditions are the same as those for the forma- 

 tion of hemoglobin from hemoglobin in the presence or absence of 

 reducing substances (cf. Chapter VIII). 



There is no need to assume that specific enzymes play a role in 

 the oxidation of hemoglobin to choleglobin and bile pigments; hemo- 

 globin is rather the catalyst of its own destruction. Enzyme systems 

 may play a role as hydrogen donors similar to that of ascorbic acid 

 (cf. Sections 4.4.4. and 4.4.5.); the enzyme systems which reduce 

 hemiglobin to hemoglobin in the erythrocyte (cf. Chapter XI, Section 

 4.) do not form choleglobin, but the reduction of hemoglobin may be 

 of importance for the formation of choleglobin. 



4.4.2. Stroma Factor. In a solution of hemolyzed red cells chole- 

 globin formation proceeds at only one third of the rate observed in 

 destromatized oxyhemoglobin solutions of the same concentration 

 (1710). The stroma thus contains a factor which protects hemo- 

 globin.* In intact red cells choleglobin is formed very slowly if at all. 

 Erythrocytes containing choleglobin can best be prepared if a small 

 hypotonicity is used in conjunction with a large ascorbic acid con- 

 centration (Lemberg and Callaghan, 169^). Such cells are unstable 



* Recent experiments (1699) indicate that the rapid choleglobin formation in acid- 

 destromatized hemolyzates of mammalian red cells can be partly explained by a com- 

 bination of lowered catalase content and increased rate of catalase destruction. A 

 stroma effect can, however, also be observed in duck erythrocytes which do not contain 

 catalase. Oxyhemoglobin solutions, exposed to a pH of 5 or below and reneutralized, 

 are thereby sensitized toward ascorbic acid + oxygen, whereas no such sensitization 

 was found on acidification of oxvgen-free hemoglobin solutions (cf. also Vladimirov 

 and Kolotilova, 289.3a). 



