Bile pigment formation in vitro from haematin and other haem derivatives.

Lemberg (1935) first showed that the green haemin of Warburg & Negelein (1930), on treatment with methanolic hydrogen chloride, gave a ferric salt of a biliverdin ester from which the ester of biliverdin itself could be obtained by shaking the chloroform solution of the salt with water. Lemberg concluded that green haemin possessed the structure of a verdohaemochromogen, i.e. it possessed an open bile pigment structure maintained as a ring by the central iron atom to which pyridine was still coordinated. The pyridine-free material (verdohaematin) on reduction with sodium amalgam gave a urobilinoid pigment with an intense Ehrlich reaction, and showed urobilin-like behaviour with zinc acetate and iodine. Condensation with ammonia reclosed the ring to produce an iron azaporphyrin with characteristic Soret band (Lemberg, 1943). Lemberg, Cortis-Jones & Norrie (1938) and Lemberg, Legge & Lockwood (1938) made detailed studies of the kinetics of the coupled oxidation of pyridine haemochromogen and ascorbic acid which leads to the formation of verdohaemochromogen. In vitro bile pigment formation from haemoglobin was describedby Lemberg, Legge & Lockwood (1939, 1941 a, b) and Lemberg, Lockwood & Legge (1941). These workers obtained evidence of an intermediate which they named choleglobin. In this compound the porphyrin ring had been opened, since the Soret band was absent; treatment of the mixture containing choleglobin with 66% acetic acid liberated two bile pigments, biliverdin and bilipurpurin. Simultaneously with the formation of the bile pigments, the choleglobin absorption at 628-630m,. disappeared. The yield of bile pigments could be correlated with intensity of the 628 mp. band. Fischer and his colleagues independently carried out researches in this field during the years 1938-42. As starting material the methyl esters ofsymmetrical ferroporphyrins were selected, since they offered a better opportunity for isolation of crystalline intermediates. The structure of the products was thus independent of the nature of the methene bridge disrupted, and methyl esters are most readily crystallizable. The oxidizing agents were hydrogen peroxide (Libowitzky, 1940), or molecular oxygen in presence of L-ascorbic acid (Fischer & Libowitzky,