486 X. BILE PIGMENT FORMATION, ETC. 



found without hem/globin being observed spectroscopically. Hem/globin, 

 which forms such a percentage of the blood pigment, should be readily found 

 in the spectroscope. In normal human blood its concentration is less than 

 1% of the total blood pigment (cf. Chapter XI, Section ;5.). 



If the differences between carbon monoxide capacities before and after 

 reduction can be confirmed, it appears unlikely that they can be due to 

 hem/globin; a part at least must be due to the presence of choleglobin or 

 similar compounds.* ^ 



6. PSEUDOHEMOGLOBIN AND CRUORALBIN 

 6.1. Pseudohemoglobin 



Green compounds with absorption spectra very similar to those of 

 choleglobin were obtained by Barkan and Schales (161,163) by 

 treating oxyhemoglobin or hemolyzed red cells with hydrogen peroxide 

 in the presence of cyanide; rather large concentrations of cyanide 

 (0.3 31) and hydrogen peroxide (about 0.1 M) were used in this 

 reaction, and alkalinity of the cyanide was not neutralized by buffer- 

 ing. The green solution had an absorption band at 617-620 m/x 

 which was shifted by carbon monoxide to 625.5 mju. By incubation 

 with 0.1 A^ hydrochloric acid, 15.6% of the iron could be removed, 

 while with carbon monoxide only 2% was detached. This compound 

 was termed "pseudohemoglobin," on the assumption that the por- 

 phyrin ring had been opened (cf. pseudouric acid, although in this 

 case the opening of the ring is due to hydrolysis, not to oxidation). 



Upon dialysis or on allowing the compound to stand, the properties were 

 found to be somewhat altered; 9% of its iron could now be detached with or 

 without carbon monoxide; carbon monoxide also no longer shifted the absorp- 

 tion band of the reduced compound in the presence of cyanide. The authors 

 attributed this to an alteration of "pseudohemoglobin" to "pseudohemo- 

 chromogen." 



Lemberg and co-workers {1709) sho^Ved that under the conditions used 

 by Barkan the globin is denatured by the alkalinity of the cyanide; on 

 dialysis the "pseudohemoglobin" precipitates completely. They failed to 

 confirm Barkan's spectroscopic observations; provided that pH and cyanide 

 concentration were identical, the green pigment behaved in the same way 

 toward carbon monoxide before and after dialysis. By buffering the cyanide 

 they obtained a pseudohemoglobin, soluble at neutral pH, which showed an 

 absorption band at 630 mju after reduction with dithionite. From these 

 experiments they concluded that pseudohemoglobin was a choleheme deriva- 



* According to Van Slyke and co-workers (357 ^a) the average difference between 

 the carbon monoxide capacities of normal human blood before and after reduction 

 was 1.3% of the total, whereas hemtglobin was only 0.4%. The difference, however, 

 decreased on standing of the blood more than the 0.4% due to the hemtglobin. 



