Modification of the Secondary Structure of Haemoprotein Molecules 89 



by the addition of lauryl sulphate was replaced by cyanide in a reaction 

 following the second order type (Fig. 7 (II)). 



In other words, the protein part of methaemoglobin seems to be modified 

 more profoundly by lauryl sulphate so as to allow even the second globin-N 

 to be replaced by cyanide. 



In an electrophoretic study (Tsushima and Kawai, 1956) of the products of 

 ferrihaemochrome formation from methaemoglobin with lauryl sulphate, two 

 components were found which had electromobilities different from that of 

 methaemoglobin. There was also a trace of another fraction which seemed to 

 be lauryl sulphate. By the addition of lauryl sulphate to methaemoglobin, 

 complex I appeared first. This complex disappeared after increased addition 

 of lauryl sulphate and instead there appeared a new complex, II. After the 

 addition of excess lauryl sulphate, only complex II remained observable. By 

 the comparison of the electrophoretic data and the spectroscopic observations 

 at various concentrations of lauryl sulphate, it was found that the process of 

 ferrihaemochrome formation corresponded to the formation of complex I, 

 and the ferrihaemochrome formation was completed at the stage of complex 

 II formation. As mentioned previously, at the stage of complex II both of two 

 globin-N's of the ferrihaemochrome can be replaced by cyanide. From the 

 foregoing results, it is assumed that the combining affinity of haem to protein 

 is affected to various degrees by the structural modification of the protein 

 part. 



Haemochrome Formation of Alkali-denatured Proteins with Haem 



With respect to the problem of haemoprotein structure and function, it is 

 desired to know to what group of the protein moiety the haem is attached. 

 One well-known approach to this problem is to digest, for instance, cyto- 

 chrome c and to isolate and analyse the haem-bound peptide. However, it is 

 also well known that haem or haematin can co-ordinate with various nitrogen 

 bases including amino acids. In this connexion, we have tried to find how 

 many haems can actually be bound to the completely unfolded protein 

 (Kajita, Uchimura, Mizutani, Kikuchi and Kaziro, 1959). 



The protein was denatured by 1 % NaOH and increasing amounts of 

 haematin were added to the denatured protein. The binding of haem with 

 the protein was measured by means of haemochrome formation after 

 reduction by sodium dithionite. Figure 8 shows a typical experiment which 

 was done with haemoglobin as the protein. With increase of haem added, the 

 optical density at 558 m/t of alkali-denatured globin haemochrome increases 

 and finally the slope of the increase in optical density becomes parallel to 

 that of the blank without protein. 



The number of haems which were bound to the protein and formed haemo- 

 chrome can be calculated tentatively by the extrapolation of the two linear 

 parts of the observed curve. The number of haem-binding groups of the 



