Modification of the Secondary Structure of Haemoprotein Molecules 91 



In other words, the affinity for haem of nitrogen of amino acids appears to 

 increase when amino acids are integrated into larger polypeptides. 



This was confirmed by our experiments with the digested protein molecule. 

 Haemoglobin was digested with pepsin and samples of the digestion mixture 

 were taken at different time intervals and tested for their haem binding 

 affinity. 



The results are shown in Fig. 9. The haem-binding affinity of the digested 

 haemoglobin decreases sharply following the time course of the digestion. 

 Also the time curve of the decrease of the haem-binding affinity is almost 



% 



10 20 30 40 



60 70 80 90 



Fig. 9. Effect of pepsin digestion on the haem-binding affinity of haemoglobin. 

 ( — o — ): per cent digestion of haemoglobin (measured by Folin test); (-•-): per 

 cent decrease of the haem-binding affinity. Haemoglobin was digested by pepsin 



at pH 2-6, 30°C. 



parallel with that of the digestion as judged by the intensity of the Folin 

 reaction. Similar results were obtained also with trypsin-digested cytochrome 

 c. These results suggest that the stability of a haemoprotein as a complex of 

 haem and native apoprotein depends principally upon the haem being bound 

 to a sufficiently large peptide molecule. The secondary structure of the 

 protein moiety may also contribute to the stabiHty as well as to the specificity 

 of individual haemoproteins. 



In this type of experiment, cytochrome c is of special interest because it is 

 very resistant to higher pH. As shown in Fig. 10, slopes of the initial part of 

 curves of the optical density change are different according to the difference 

 in pH of the reaction mixture. 



Taking the haem binding groups obtained at the highest pH to be 100%, 

 the percentage of haem binding groups which is available at each pH was 

 calculated and plotted against pH. The results are shown in Fig. 11. The 

 curve coincides approximately with the theoretical curve of a first order 

 reaction. We do not know, however, whether the curve of Fig. 11 is a reflec- 

 tion of the extent of unfolding of the cytochrome c molecule at different pH 



H.E. — ^VOL. I — H 



