460 X. BILE PIGMENT FORMATION, ETC. 



split off water from the side chains of hematobiliverdin by the methods used 

 for preparing protoporphyrin from hematoporphyrin, but partial success 

 was achieved. Tliis difficulty was later also observed by Stier (2666). No 

 isomeric protobiliverdin esters could be detected in the mother liquor. 



The mesobiliverdin dimethyl ester obtained in the same way from mesohemo- 

 chrome had a melting point of 218-219° and gave no depression with the 

 ester (m.p. 220°) prepared from mesobilirubin; similarly, the ferrichloride 

 of the ester obtained from mesohemochrome melted at 261° and gave no 

 depression witli the ester prepared from mesobilirubin (265°). In this case, 

 however, there was evidence for the presence of isomerides in the mother 

 liquor from which an ester of lower melting point (179-180°) was obtained. 



These experiments show that the methene bridge a is removed 

 preferentially. No evidence was found that the ester formed from 

 protohemochrome was a mixture of isomerides, whereas with meso- 

 hemochrome there was evidence that other isomerides were formed. 



In the case of biliverdin ester (m.p. 218°) obtained from hemoglobin 

 by coupled oxidation with ascorbic acid, Lemberg and collaborators 

 (1707) again found no evidence for the formation of a mixture of 

 isomerides. 



Although there is thus some evidence that methene groups other 

 than the group a are attacked if mesohemochrome is oxidized, such 

 evidence is absent for the oxidation of protohemochrome. 



Whether or not, in the case of protoheme, the group a alone is attacked 

 in the reaction, tliere is no evidence whatsoever for a greater specificity of 

 the bile pigment formation iji vivo than in vitro. During the isolation of 

 bilirubin from gall stones or bile, a large proportion of the compound remains 

 in the mother liquors and more easily soluble isomerides may thus escape 

 detection. In other words, selection of the most readily crystallizable bile 

 pigments may indicate a specificity of the removal of the a group in vivo as 

 well as in vitro, which in fact may not be complete. It may or may not be 

 significant that the melting points of mesobilene-(b) hydrochloride prepa- 

 rations obtained from natural bilirubin (3001) vary widely and are far below 

 that of the synthetic substance of Siedel (2550). 



Nevertheless, we believe that there is a directive influence which 

 makes the methene group a especially likely to undergo oxidative 

 removal. This can be partly ascribed to the carboxylic acid groups 

 on the opposite side of the molecule which give it a polar character 

 and make the methene group furthest away from them the most 

 readily attacked. The neighborhood of a vinyl group may enhance 

 this effect. It is also probable that the specific effect is larger for 

 hemoglobin than for hemochrome. 



