84 K. Kaziro and K. Tsushima 



(from 0-5 to 50m), whereas the sigmoid coefficient remained constant at 

 concentrations of urea up to 2-0 m. 



It is apparent that the affinity of haemoglobin for EIC is very sensitive to 

 even a shght modification by urea of the haemoglobin structure, whereas the 

 haem-haem interaction is more resistant. The observed alteration of the 

 ElC-combining affinity may be the result of the loosening of hydrogen bonds 

 in the protein molecule. The secondary structure of a protein molecule thus 

 modified may well be variable according to the extent of disruption of 

 hydrogen bonds. The disruption seems to occur uniformly over the haemo- 

 globin molecules as judged from the symmetry of the sigmoid curves obtained 

 experimentally. It is interesting to note in the experiments with urea that the 

 binding affinity of haemoglobin with EIC was not affected by pH changes 

 within 5-3 to 9-3 (Fig. 3 (II)). Figure 3 shows the relationship between Y^q 

 and pH in the presence and absence of urea. The results suggest that the Bohr 

 effect is associated only with the intact haemoglobin molecule. The linkage 

 of haem iron and acid groups of the haemoglobin molecule, which presumably 

 is responsible for the Bohr effect, may be disrupted in the denaturation of the 

 globin moiety by urea. 



Summarizing the experimental results so far presented we may conclude 

 that the binding affinity of haemoglobin with oxygen, the haem-haem inter- 

 action, and the Bohr effect of haemoglobin are all intimately connected with 

 the secondary structure of the protein part, although these functions appear 

 to be independent of each other. Particularly, the Bohr effect seems to be 

 associated with a highly specific structure of the protein molecule. 



The Effect of Modification of the Protein Moieties of Haemoglobin and 

 Cytochrome c on Their Catalytic Activities as Oxidases 



In order to obtain further information as to how far the specific functions 

 of individual haematin enzymes are dependent upon their protein structure, 

 we have investigated the alteration of the proper functionings of haemopro- 

 teins after the modification of their protein parts. This approach could provide 

 a new clue to the problem. Haemoglobin, myoglobin and cytochrome c were 

 the materials of our study along this line. 



As reagents which cause the modification or disintegration of the secondary 

 structure of the protein molecule, we have used various carboxylic acid salts 

 such as benzoate, salicylate, laurate and palmitate. In some cases, we have 

 tried sodium lauryl sulphate also. 



As has been reported by Anson (1929, 1934) and Holden (1947), addition 

 of these salts to methaemoglobin results in the spectral change of methaemo- 

 globin to ferrihaemochrome indicating alteration of the secondary structure 

 of the molecule. We have observed also that the addition of these salts 

 enhanced the autoxidation of oxyhaemoglobin and subsequently increased 

 its oxidase activity (Tsushima, 1954b; Kikuchi and Tomimura, 1954). 



