232 VI. HEMOGLOBIN 



portion of the hemoglobin molecule. The change from ionic to covalent 

 bonds on oxygenation of hemoglobin is assumed to be due to a secondary 

 alteration of the linkages binding the heme to the protein when the oxygen 

 combines. Now, although the existence of Hill's native globin hemochrome 

 (c/. Section 4.3.3.1.) indicates that nitrogenous groups capable of forming 

 covalent bonds with the iron atom lie in proximity to the heme, the spectrum 

 of oxyhemoglobin is not that of a hemochrome and Holden's suggestion 

 appears most unlikely. 



3.2.2. Evidence That Heme Iron Combines with Imidazoles 



3.2.2.1. Origin of the Imidazole Hypothesis. The recognition of 

 the similarity between denatured globin hemochrome and the hemo- 

 chromes containing simple bases (Chapter V) drew attention to the 

 importance of the groups responsible for hemochrome formation in 

 denatured globin. In v4ew of the large amount of histidine in globin, 

 Kiister (1611) and later Langenbeck [161^2,161^3) suggested that 

 histidine imidazoles were involved in the linkage of heme in hemo- 

 globin. Haurowitz {1165) opposed this hypothesis because of the low 

 affinity of histidine for heme; but histidine is present in peptide 

 linkage in the protein and, though some histidine peptides have been 

 prepared {1052), their affinity for heme has not been measured. 



The histidine content, however, varies considerably among hemoglobins. 

 In chlorocruorin (Chapter VII), an invertebrate oxygen carrier, about 2.5 

 moles of histidine are present per mole of heme, while Vickery gives 33 moles 

 of histidine per mole (four hemes) of hemoglobin. While globin will combine 

 with only one heme for an equivalent molecular weight of 16,700 {1282), 

 denatured globin will combine with as many as six hemes per equivalent 

 {1311,1322,3105), i.e., more than the maximum possible if each heme binds 

 two histidines. Holden and Freeman {1322) found that the ability of dena- 

 tured ox globin to form hemochromes was diminished by treatment with 

 nitrous acid, a procedure which leaves imidazoles unaffected, and concluded 

 that imidazole was not solely responsible for hemochrome formation. Cyto- 

 chrome c, which contains only three histidines, is also able to combine with 

 additional hemes after denaturation {3165). The imidazole groups are 

 therefore not the only groups in protein with which the heme iron can 

 combine to form hemochromes. 



Conant's discussion of the linkage between heme and globin in 

 1933 {-^71) marked an important advance, since he took into account 

 the earlier work {cf. Section 3.1.), in which oxygenation had been 

 shown to affect the properties of the protein. In Haurowitz's dis- 

 cussion of the linkage from the point of view of the Werner coordi- 

 nation theory, the sixth coordination valency of the iron in hemo- 

 globin was occupied by a molecule of water, which was displaced on 

 oxygenation. Conant considered that in hemoglobin the heme might 



