LINKAGE OF PROTEIN TO PROSTHETIC GROUP 231 



evidence as to the nature of the groups in the protein, they were 

 called "hemaffine" groups. 



Attempts have been made to degrade hemoglobin in the hope that a 

 fragment could be isolated in which the original linkage between the heme 

 and the hemaflBne groups could be more readily investigated than in the 

 original protein. Starting with the work of von Zeynek (3178) in 1906, all 

 such attempts have been unsuccessful. Enzymic methods have generally 

 been used for this since all other treatments detach the heme completely. 

 Thus, Haurowitz {1160) subjected hemoglobin to tryptic digestion and 

 isolated a "hemin proteose." He was unable, however, to detach hematin 

 from this product, even by treatment with hydrobromic acid in acetic acid. 

 The original linkage in the native protein was, therefore, no longer present 

 in the pigment. Ross (^-544) and Ross and Turner {23Jto) extended this 

 approach with the investigation of the pancreatic digestion of carboxyhemo- 

 globin. The outcome of their work was, however, as indecisive as that of 

 Haurowitz so far as the original linkage was concerned. 



Little support has been given to the view that the prosthetic group is 

 bound to the protein by peptide or ester linkages; the ease with which the 

 prosthetic group may be removed or recombined with the protein {cf. Section 

 4.3.) makes such linkages unlikely. Most of the work has suggested labile 

 coordinate bonds between the iron atom rnd the protein or some type of 

 feeble linkage between the protein and other parts of the prosthetic group. 

 These possibilities will be discussed separately. 



3.2. The Linkage of Heme Iron to Globin 



3.2.1. Magnetochemical Evidence. The heme iron is involved 

 not only in the attachment of heme to globin in hemoglobin, but also 

 in the combination of hemoglobin with other molecules such as oxygen 

 or carbon monoxide. While Haurowitz and Waelsch (1177) formu- 

 lated the derivatives of hemoglobin as coordination compounds in 

 1929, the most direct evidence for this assumption has come later 

 from the magnetochemical investigations of Pauling and co-workers. 

 By combination with oxygen or carbon monoxide, the paramagnetic 

 hemoglobin (with four unpaired electrons in its iron atom) is trans- 

 formed to a diamagnetic compound (no unpaired electrons) ; the para- 

 magnetic oxygen molecule also loses its unpaired electrons in its 

 combination with hemoglobin. Such changes in the electronic con- 

 figuration of the iron atom can be explained only if the globin as well 

 as the molecules combining with hemoglobin are attached to the 

 heme iron. 



In order to account for some of the differences between oxyhemoglobin 

 and carboxyhemoglobin, Holden (1317) has suggested that, in the former 

 case, the oxygen is not combined with the iron atom but with some other 



