Modification of the Secondary Structure of Haemoprotein Molecules 87 



appearance of CO-binding affinity by the modification of the cytochrome c 

 molecule, because we know that the molecular size of CO is almost the same 

 as that of O2 and generally CO has strong affinity for many haem compounds. 

 The experimental facts may, in turn, be evidence for the significance of the 



100 



50 E 



Benzoote, M 



Fig. 6. Effect of benzoate on the oxidase activity of cytochrome c. The concen- 

 trations of cytochrome c and ascorbic acid were 3-31 x 10~^ m and 1-5 x 10"^ M, 

 respectively. The concentration of benzoate was varied from to 1-75 m. The 

 measurements of CO-compound formation were made spectrophotometrically at 

 550 m/i. The measurement of oxidase activity and the expression of ordinate are 

 the same as those of Fig. 4. 



secondary structure of the molecule as the determining factor of the specificity 

 of haemoproteins in general. 



Binding Affinity of Haem with Proteins 



Thus far, our experiments in section (1) and (2) have been concerned with 

 the relationship between the function and the secondary structure of haemo- 

 proteins. Now, we wish to see how far the modification of the secondary 

 structure of the protein moiety can proceed. As an approach to the study of 

 this problem, we examined the affinity of the protein moiety for haem after 

 controlled alteration of the protein part (Tsushima, 1956a). As mentioned 

 previously, when benzoate or lauryl sulphate was added to a methaemoglobin 

 solution, a ferrihaemochrome spectrum was seen at a definite concentration 

 range of the agent added. The nature and the extent of modification of the 

 protein structure which led to the ferrihaemochrome spectrum is unclear as 

 yet. However, it is certain that some nitrogen base of the unfolded protein 

 is bound to the 6th co-ordination position of the haem-iron. As a matter of 

 fact, the globin-N in the ferrihaemochrome structure can readily be replaced 

 by cyanide as well as by azide. 



