230 VI. HEMOGLOBIN 



3. LINKAGE OF PROTEIN TO PROSTHETIC GROUP 

 3.1. Introduction 



Hemoglobin was the first conjugated protein investigated, and the 

 mode of linkage of the prosthetic group to the protein, which is of 

 equal interest for other hemoproteins, was first studied with hemo- 

 globin. Progress may be divided into that made up to the mid-1920's 

 and that made since, the dividing line being established by many 

 important advances made at the beginning of the latter period. The 

 molecular weight of hemoglobin was satisfactorily determined in 

 1925, while, in the same year, progress in spectroscopy cleared up the 

 confusion between denatured globin hemichrome (kathemoglobin) 

 and oxyhemoglobin (65,lJf.75). In 1926, native globin was first 

 prepared and hemoglobins were synthesized which carried prosthetic 

 groups other than protoheme {1282). In 1928, the structure and 

 shape of hemin was definitely established, and it became possible to 

 envisage some of the steric considerations involved in the linkage. 



In spite of lack of knowledge of these facts, the early workers 

 made considerable progress. That hemoglobin could be split by 

 acids was well known in the latter part of the nineteenth century 

 and this fact, together with the large basic amino acid content of 

 hemoglobin, suggested that the heme was bound to basic groups. 

 Laidlaw's finding in 1904 {1632) that the iron was more readily split 

 from hemoglobin than from oxyhemoglobin showed that the stability 

 of the prosthetic group varied in different derivatives. The earlier 

 work also clearly established the complexity of the interaction between 

 oxygen, prosthetic group, and protein, which must be explained by 

 any adequate hypothesis for the linkage. Bohr, Hasselbach, and 

 Krogh {309a) showed in 1904 that carbon dioxide decreased the 

 affinity of hemoglobin for oxygen {cf. Section 5.), while in 1914 

 Christiansen, Douglas, and Haldane {Ifli-2a) showed the existence of 

 the converse effect, oxygenation diminishing the amount of carbon 

 dioxide bound by the blood. Later work {cf. Roughton, 2362) has 

 shown that these effects are due both to the increased acidity of the 

 oxyhemoglobin and to the diminished amount of carbon dioxide 

 bound as carbhemoglobin. 



At various times the prosthetic group has been considered as 

 forming a molecular compound with the globin and as being linked 

 through peptide, salt, or ester linkages formed by the heme carboxyl 

 groups and groups in the globin or through coordinate linkage from 

 the iron to some group in the protein. In the absence of definite 



