ROLE OF HEMATIN SIDE CHAINS IN LINKAGE 239 



groups have been shown to form a carbamino compound according 

 to the equation: 



RN H2 + CO2 -^ RNHCOOH 



Imidazole as well as various other groups has been tested and found 

 to be unable to form carbamino compounds. As a consequence of 

 this fact Houghton has examined the evidence for the imidazole 

 hypothesis and has made a number of criticisms. In addition to 

 suggesting the possibility of certain technical errors in Wyman's 

 technique, which might lead to an accuracy in his heats of reaction of 

 only ± 1000 cal., Roughton points out that the heats of dissociation 

 of hydrogen ions from horse hemoglobin in the pH range 6 to 8 are 

 some 2000 cal. lower than the value found with ox hemoglobin, the 

 latter value suggesting that both imidazole and amino groups are 

 involved in buffering in this species. 



Further he criticizes the lack of rigor in identification of a particular 

 pK value in a protein with a particular dissociating group. Roughton 

 points out that glycylglycine is able to form thirty times as many 

 carbamino groups as glycine at pH 8, in agreement with the fact 

 that the pK value of the free amino in the peptide lies about two pH 

 units nearer neutrality than in the free amino acid. On the other 

 hand, replacement of one of the postulated heme-linked imidazoles 

 by a glycyl residue, or by the terminal amino group of a lysyl residue 

 will necessitate a re-examination of the mechanism by which the entry 

 of oxygen affects the dissociation of hydrogen from the oxylabile 

 groups. This problem is referred to again in Section 8. 



3.3. Role of Hematin Side Chains in Linkage 



3.3.1. Introduction. A number of the reactions of globin can be interpreted in 

 terms of the linkage between the iron atom in the heme and the "hemaffine" 

 groups in the protein. The evidence for linkage through iron, however, does 

 not exclude the possibility that the side chains may be involved as well. 



The ease with which the prosthetic group may be removed makes it 

 unlikely that ester or peptide linkages are present. Only linkages of an 

 electrostatic nature seem possible. These may be in the nature of weak van 

 der Waals forces, or linkages involving the carboxyls or perhaps the vinyl 

 side chains in the hematin. The latter possibility is excluded by consideration 

 of synthetic hemoglobins. 



3.3.2. Synthetic Hemoglobins. Hill and Holden {1282) prepared meso- 

 hemoglobin and hematohemoglobin. Both were able to combine reversibly 

 with oxygen. Warburg and Negelein (29o4-) prepared hemoglobins from 



